Link to Odell’s “How to Do Nothing”

eLife assessment

This study provides valuable insights into the specificity and promiscuity of toxic effector and immunity protein pairs. While the work is improved over a previous version, there are still some questions regarding the methodology used to draw certain conclusions, rendering the study somewhat incomplete. Nevertheless, this work will likely be of interest to microbiologists and biochemists working with toxin-antitoxin systems and effector-immunity proteins.

Reviewer #3 (Public Review):

Summary:

The authors discovered that the RdnE effector possesses DNase activity, and in competition, P. mirabilis having RdnE outcompetes the null strain. Additionally, they presented evidence that the RdnI immunity protein binds to RdnE, suppressing its toxicity. Interestingly, the authors demonstrated that the RdnI homolog from a different phylum (i.e., Actinomycetota) provides cross-species protection against RdnE injected from P. mirabilis, despite the limited identity between the immunity sequences. Finally, using metagenomic data from human-associated microbiomes, the authors provided bioinformatic evidence that the rdnE/rdnI gene pair is widespread and present in individual microbiomes. Overall, the discovery of broad protection by non-cognate immunity is intriguing, although not necessarily surprising in retrospect, considering the prolonged period during which Earth was a microbial battlefield/paradise.

Strengths:

The authors presented a strong rationale in the manuscript and characterized the molecular mechanism of the RdnE effector both in vitro and in the heterologous expression model. The utilization of the bacterial two-hybrid system, along with the competition assays, to study the protective action of RdnI immunity is informative. Furthermore, the authors conducted bioinformatic analyses throughout the manuscript, examining the primary sequence, predicted structural, and metagenomic levels, which significantly underscore the significance and importance of the EI pair.

Weaknesses:

(1) The interaction between RdnI and RdnE appears to be complex and requires further investigation. The manuscript's data does not conclusively explain how RdnI provides a "promiscuous" immunity function, particularly regarding the RdnI mutant/chimera derivatives. The lack of protection observed in these cases might be attributed to other factors, such as a decrease in protein expression levels or misfolding of the proteins. Additionally, the transient nature of the binding interaction could be insufficient to offer effective defenses.<br /> (2) The results from the mixed population competition would benefit from quantitative analysis. The swarm competition assays only yield binary outcomes (Yes or No), limiting the ability to obtain more detailed insights from the data.<br /> (3) The discovery of cross-species protection is solely evident in the heterologous expression-competition model. It remains uncertain whether this is an isolated occurrence or a common characteristic of RdnI immunity proteins across various scenarios. Further investigations are necessary to determine the generality of this behavior.

Reviewer #4 (Public Review):

Summary:

Knecht et al. elucidate a Type VI Secretion System (T6SS) effector-immunity pair in Proteus mirabilis. They demonstrate that the effector protein RdnE exhibits DNase activity in vitro and induces toxicity when ectopically expressed in cells, the latter being neutralized by the cognate immunity protein RdnI. The authors identify major regions within RdnI necessary for the interaction and neutralization of RdnE. Notably, they report cross-talk where both cognate and non-cognate RdnI proteins can neutralize RdnE, mitigating its fitness advantage in bacterial co-swarm assays. A comprehensive metagenomic analysis revealed an abundance of rdnI over rdnE genes in most gut samples, suggesting a potential role of rdnI in providing a fitness advantage against bacteria encoding for RdnE effector.

Strengths:

The authors successfully combined biochemical and microbiological experiments with bioinformatics analysis to advance the understanding of the T6SS-mediated population dynamics in bacteria. The co-swarm functional assay is of particular interest as it demonstrates how bacterial strains carrying only rdnI immunity genes could potentially compete in the same niche with other species armed with toxic rdnE effector genes. The manuscript is well-written, and the figures are self-explanatory.

Weaknesses:

(1) How would the authors explain the discrepancy observed in Figure 4 G and Figure 4 S3 B where two RdnI proteins from Prevotella and Pseudomonas genera do not bind to RdnE_Proteus in BACTH, whereas they co-elute with a RdnE_Proteus-FLAG with efficiency comparable to the cross-neutralizing RdnI_Rothia? Similarly, the interaction results obtained in BACTH with RdnI truncate (Figure 4E) or chimeric RdnI (Figure 4I, lane 4) could be a result of an overexpressed T18-fusion variant.<br /> Alternative in vitro protein binding assay would be beneficial.

(2) Based on the bioinformatic analysis the Rothia and Prevotella species harboring rdnE/I genes co-occurred in 5% of metagenomes tested, suggesting that these bacteria could come into contact. The manuscript would benefit greatly if authors demonstrated that RdnI proteins from Rothia or Prevotella could cross-neutralize its own and its 'neighbor' RdnE effectors, for example in an E. coli viability assay. The cross-neutralizing co-swarming results (Figure 4F) could also be further validated in viability assay as shown in Figure 2 S1.

(3) Little is known about whether RdnE is delivered via T6SS as a full-length protein or as the shorter C-terminal fragment. There is a possibility that immunity proteins could recognize RdnE regions beyond the C-terminal 138 amino acids that authors used in their in vitro assays.

Reviewer #5 (Public Review):

This work investigates a T6SS effector-immunity pair from Proteus mirabilis. The authors make several interesting claims, particularly regarding the mechanism of effector inhibition by the immunity protein. However, it appears that these claims are not fully supported by the evidence provided.

I have read the revised manuscript, the public reviews, and the authors' updated responses to these reviews. In my opinion, the concerns raised by the reviewers remain relevant even after the authors' revisions. Since previous reviews have excellently described the strengths and weaknesses of this work, I will focus on my major concerns:

(1) The authors describe RdnE-RdnI, a T6SS effector-immunity pair from Proteus mirabilis. RdnE is actually the C-terminal domain of IdrD, a 1581-amino-acid protein containing PAAR and RHS domains. This work does not provide evidence for T6SS-dependent secretion of the effector, instead supplying references to previous works.

(2) While the authors claim the function of the RdnE domain is unknown, it was previously shown to be evolutionarily related to PoNe and TseV, both of which are known DNA nucleases. Although the authors cite the relevant references, they do not clearly disclose this information.

(3) The authors claim that RdnE contains two different domains: the first is the PD-(D/E)XK domain, and the second, referred to as "region 2," follows it. Unfortunately, no structural evidence is provided to support this claim, not even a predicted model demonstrating that these are indeed separate domains.

(4) One of the major claims made in this work is that RdnI binding to RdnE is not sufficient for RdnE inhibition, suggesting a more sophisticated mechanism. The authors base this theory on differences between the ability of RdnI to bind RdnE (shown using bacterial two-hybrid assays) and the ability to protect against RdnE toxicity in swarm competition assays. Specifically, they show that the first 85 amino acids of RdnI bind to the short RdnE domain in the bacterial two-hybrid assay but do not protect against the full-length effector in the swarm competition assay. They also demonstrate that performing seven mutations in conserved residues in RdnE or replacing parts of RdnI with parts from other RdnI homologs leads to the same phenomenon.

While these findings are interesting and even intriguing, in my opinion, the current evidence does not support their theory. A simple explanation for the differences between the assays is that while the N-terminal domain of RdnI is sufficient for binding to RdnE, inhibition of the active site of RdnE requires binding of a second domain to RdnE. In that sense, it should be noted that while the authors use co-IP assays to show the interaction between RdnE and full-length RdnI, they do not use it to show the interaction between RdnE and the first 85 amino acids of RdnI.

(5) The authors claim that a "conserved motif" within RdnI plays a role in the inhibition of RdnE. To investigate this, they replace this motif with sequences from several RdnI homologs, demonstrating that in one case, it is possible to exchange these conserved motifs between RdnI homologs that inhibit Proteus RdnE. However, they also show that even if the conserved motif is taken from an RdnI homolog that cannot inhibit Proteus RdnE, the hybrid protein can still protect cells in a swarm competition assay. This result raises concerns regarding the relevance of this conserved motif.

(6) Lastly, regarding the theory that immunity proteins can protect against non-cognate effectors, it appears that the authors based their theory on a single case where RdnI from Rothia protected against RdnE from Proteus. In my opinion, a more thorough investigation, involving testing many homologs, is needed to substantiate this theory.

Author response:

The following is the authors’ response to the original reviews.

eLife assessment

This work presents valuable information about the specificity and promiscuity of toxic effector and immunity protein pairs. The evidence supporting the claims of the authors is currently incomplete, as there is concern about the methodology used to analyze protein interactions, which did not take potential differences in expression levels, protein folding, and/or transient interaction into account. Other methods to measure the strength of interactions and structural predictions would improve the study. The work will be of interest to microbiologists and biochemists working with toxin-antitoxin and effector-immunity proteins.

We thank the reviewers for considering this manuscript. We agree that this manuscript provides a valuable and cross-discipline introduction to new EI pair protein families where we focus on the EI pair’s flexibility and impacts on community structure. As such, we believe we have provided a solid foundation for future studies to examine non-cognate interactions and their possible effects on microbial communities. This, by definition, leaves some areas “incomplete” and, therefore, open for further investigations. While the methods we show do consider potential differences in binding assays, we have more explicitly addressed how “expression, protein folding, and/or transient binding” may play into this expanded EI pair model. We have also tempered the discussion of the proposed model, while also clearly highlighting other published evidence of non-cognate binding interactions between effector and immunity proteins. We have responded to the reviewers’ public comments (italicized below). 

In this revised manuscript, we have updated the main text, particularly the Discussion section, to include more careful language, explain past research better, and add new references to works showing non-cognate immunity proteins protecting against effectors in other systems. We have also updated the supplemental files with more analyses; the relevant procedures are in the Materials and Methods.

Public Reviews:

Note: Reviewer 1, who appeared to focus on a subset of the manuscript rather than the whole, based their comments on several inaccuracies, which we discuss below. We found the tone in this reviewer's comments to be, at times, inappropriate, e.g., using "harsh" and "simply too drastic" to imply that common structure-function analyses were outside of the field-standard methods. We also note that the reviewer took a somewhat atypical step in reviewing this manuscript by running and analyzing the potential protein-complex data in AlphaFold2 but did not discuss areas of low confidence within that model that may contradict their conclusions. We are concerned their approach muddled valid scientific criticisms with problematic conclusions.

Reviewer #1 (Public Review):

In this manuscript, Knecht, Sirias et al describe toxin-immunity pair from Proteus mirabilis. Their observations suggest that the immunity protein could protect against non-cognate effectors from the same family. They analyze these proteins by dissecting them into domains and constructing chimeras which leads them to the conclusion that the immunity can be promiscuous and that the binding of immunity is insufficient for protective activity.

Strengths:<br />  The manuscript is well written and the data are very well presented and could be potentially interesting. The phylogenetic analysis is well done, and provides some general insights.

Weaknesses:<br /> (1) Conclusions are mostly supported by harsh deletions and double hybrid assays. The later assays might show binding, but this method is not resolutive enough to report the binding strength. Proteins could still bind, but the binding might be weaker, transient, and out-competed by the target binding.

The phrasing of structure-function analyses as “harsh” is a bit unusual, as other research groups regularly use deletions and hybrid studies. Given the known caveats to deletion and domain substitutions, we included point-mutation analyses for both the effector and immunity proteins, as found on lines 105 - 113 and 255 - 261 in the current manuscript. These caveats are also why we coupled the in vitro binding analyses with in vivo protection experiments in two distinct experimental systems (E. coli and P. mirabilis). Based on this manuscript’s introductory analysis (where we define and characterize the genes, proteins, interactions, phylogenetics, and incidences in human microbiomes), the next apparent questions are beyond the scope of this study. Future approaches would include analyzing purified proteins from the effector (E) and immunity (I) protein families using biochemical assays, such as X-ray crystallography, circular dichroism spectroscopy, among others. 

Interestingly, most papers in the EI field do not measure EI protein affinity (Jana et al., 2019, Yadav et al., 2021). Notable exceptions are earlier colicin research (Wallis et al., 1995) and a new T6SS EI paper (Bosch et al., 2023) published as we first submitted this manuscript.

(2) While the authors have modeled the structure of toxin and immunity, the toxin-immunity complex model is missing. Such a model allows alternative, more realistic interpretation of the presented data. Firstly, the immunity protein is predicted to bind contributing to the surface all over the sequence, except the last two alpha helices (very high confidence model, iPTM>0.8). The N terminus described by the authors contributes one of the toxin-binding surfaces, but this is not the sole binding site. Most importantly, other parts of the immunity protein are predicted to interact closer to the active site (D-E-K residues). Thus, based on the AlphaFold model, the predicted mechanism of immunization remains physically blocking the active site. However, removing the N terminal part, which contributes large interaction surface will directly impact the binding strength. Hence, the toxin-immunity co-folding model suggests that proper binding of immunity, contributed by different parts of the protein, is required to stabilize the toxin-immunity complex and to achieve complete neutralization. Alternative mechanisms of neutralization might not be necessary in this case and are difficult to imagine for a DNase.

In response to the reviewer’s comment, we again reviewed the RdnE-RdnI AlphaFold2 complex predictions with the most updated version of ColabFold (1.5.2-patch with PDB100 and MMseq2) and have included them at the end of these responses [1].

However, the literature reports that computational predictions of E-I complexes often do not match experimental structural results (Hespanhol et al., 2022, Bosch et al., 2023). As such, we chose not to include the predicted cognate and non-cognate RdnE-I complexes from ColabFold (which uses AlphaFold2) and have not included this data in the revised manuscript. (It is notable that reviewer 1 found the proposed expanded model and research so interesting as to directly input and examine the AI-predicted RdnE-RdnI protein interactions in AlphaFold2.)

Discussion of the prevailing toxin-immunity complex model is in the introduction (lines 45-48) and Figure 5E. Further, there are various known mechanisms for neutralizing nucleases and other T6SS effectors, which we briefly state in the discussion (lines 359 - 361). More in-depth, these molecular mechanisms include active-site blocking (Benz et al., 2012), allosteric-site binding (Kleanthous et al., 1999 and Lu et al., 2014), enzymatic neutralization of the target (Ting et al., 2021), and structural disruption of both the active and binding sites (Bosch et al., 2023). Given this diversity of mechanisms, we did not presume to speculate on the as-of-yet unknown mechanism of RdnI protection. We have expanded discussion of these items in the revised manuscript.

(3) Dissection of a toxin into two domains is also not justified from a structural point of view, it is probably based on initial sequence analyses. The N terminus (actually previously reported as Pone domain in ref 21) is actually not a separate domain, but an integral part of the protein that is encased from both sides by the C terminal part. These parts might indeed evolve faster since they are located further from the active site and the central core of the protein. I am happy to see that the chimeric toxins are active, but regarding the conservation and neutralization, I am not surprised, that the central core of the protein fold is highly conserved. However, "deletion 2" is quite irrelevant - it deletes the central core of the protein, which is simply too drastic to draw any conclusions from such a construct - it will not fold into anything similar to an original protein, if it will fold properly at all.

The reviewer’s comment highlights why we turned to the chimera proteins to dissect the regions of RdnE (formerly IdrD-CT), as the deletions could result in misfolded proteins. (We initially examined RdnE in the years before the launch of AlphaFold2.) However, the reviewer is incorrect regarding the N-terminus of RdnE. The PoNe domain, while also a subfamily of the PD-(D/E)XK superfamily, forms a distinct clade of effectors from the PD-(D/E)XK domain in RdnE (formally IdrD-CT) as seen in Hespanhol et al., 2022; this is true for other DNase effectors as well. Many studies analyzing effectors within the PD-(D/E)XK superfamily only focus on the PD-(D/E)XK domain, removing just this domain from the context of the whole protein (Hespanhol et al., 2022; Jana et al., 2019). Of note, in RdnE, this region alone (containing the DNA-binding domain) is insufficient for DNase activity (unlike in PoNe). We have clarified this distinction in the results section of the current manuscript, visible in figure 2 .

(4) Regarding the "promiscuity" there is always a limit to how similar proteins are, hence when cross-neutralization is claimed authors should always provide sequence similarities. This similarity could also be further compared in terms of the predicted interaction surface between toxin and immunity.

Reviewer 1 points out a fundamental property of protein-protein interactions that has been isolated away from the impacts of such interactions on bacterial community structure. We have provided the whole protein alignments in figure 3 supplemental figure 3, the summary images in Figure 3D, and the protein phylogenetic trees in Figure 3C. We encourage others to consider the protein alignments as percent amino acid sequence similarity is not necessarily a good gauge for protein function and interactions. These data are publicly available on the OSF website associated with this manuscript https://osf.io/scb7z/, and we hope the community explores the data there.

In consideration of the enthusiasm to deeply dive into the primary research data, we have included the pairwise sequence identities across the entire proteins here: Proteus RdnI vs. Rothia RdnI: 23.6%; Proteus RdnI vs. Prevotella RdnI: 16.3%, Proteus RdnI vs. Pseudomonas RdnI: 14.6%; Rothia RdnI vs. Prevotella RdnI: 22.4%, Rothia RdnI vs. Pseudomonas RdnI: 17.6%; Prevotella RdnI vs. Pseudomonas RdnI: 19.5%. (As stated in response to reviewer 1 comment 2, we did not find it appropriate to make inferences based on AlphaFold2-predicted protein complexes.)

Overall, it looks more like a regular toxin-immunity couple, where some cross-reactions with homologues are possible, depending on how far the sequences have deviated. Nevertheless, taking all of the above into account, these results do not challenge toxin-immunity specificity dogma.

In this manuscript, we did not intend to dismiss the E-I specificity model but rather point out its limitations and propose an important expansion of that model that accounts for cross-protection and survival against attacks from other genera. We agree that it is commonly considered that deviations in amino acid sequence over time could result in cross-binding and protection (see lines 364-368). However, the impacts of such cross-binding on community structure, bacterial survival, and strain evolution were rarely addressed in prior literature, with exceptions such as in Zhang et al., 2013 and Bosch et al., 2023 among others. One key insight we propose and show in this manuscript is that cross-binding can be a fitness benefit in mixed communities; therefore, it could be selected for evolutionarily (lines 378-380), even potentially in host microbiomes.

Reviewer #2 (Public Review):

Summary:

The manuscript by Knecht et al entitled "Non-cognate immunity proteins provide broader defenses against interbacterial effectors in microbial communities" aims at characterizing a new type VI secretion system (T6SS) effector immunity pair using genetic and biochemical studies primarily focused on Proteus mirabilis and metagenomic analysis of human-derived data focused on Rothia and Prevotella sequences. The authors provide evidence that RdnE and RdnI of Proteus constitute an E-I pair and that the effector likely degrades nucleic acids. Further, they provide evidence that expression of non-cognate immunity derived from diverse species can provide protection against RdnE intoxication. Overall, this general line of investigation is underdeveloped in the T6SS field and conceptually appropriate for a broad audience journal. The paper is well-written and, aside from a few cases, well-cited. As detailed below however, there are several aspects of this paper where the evidence provided is somewhat insufficient to support the claims. Further, there are now at least two examples in the literature of non-cognate immunity providing protection against intoxication, one of which is not cited here (Bosch et al PMID 37345922 - the other being Ting et al 2018). In general therefore I think that the motivating concept here in this paper of overturning the predominant model of interbacterial effector-immunity cognate interactions is oversold and should be dialed back.

We agree that analyses focusing on flexible non-cognate interactions and protection are underdeveloped within the T6SS field and are not fully explored within a community structure. These ideas are rapidly growing in the field, as evidenced by the references provided by the reviewer. As stated earlier, we did not intend to overturn the prevailing model but rather have proposed an expanded model that accounts for protection against attacks from foreign genera.

Strengths:

One of the major strengths of this paper is the combination of diverse techniques including competition assays, biochemistry, and metagenomics surveys. The metagenomic analysis in particular has great potential for understanding T6SS biology in natural communities. Finally, it is clear that much new biology remains to be discovered in the realm of T6SS effectors and immunity.

Weaknesses:

The authors have not formally shown that RdnE is delivered by the T6SS. Is it the case that there are not available genetics tools for gene deletion for the BB2000 strain? If there are genetic tools available, standard assays to demonstrate T6SS-dependency would be to interrogate function via inactivation of the T6SS (e.g. by deleting tssC).

Our research group showed that the T6SS secretes RdnE (previously IdrD) in Wenren et al., 2013 (cited in lines 71-73). We later confirmed T6SS-dependent secretion by LC-MS/MS (Saak et al., 2017).  

For swarm cross-phyla competition assays (Figure 4), at what level compared to cognate immunity are the non-cognate immunity proteins being expressed? This is unclear from the methods and Figure 4 legend and should be elaborated upon. Presumably these non-cognate immunity proteins are being overexpressed. Expression level and effector-to-immunity protein stoichiometry likely matters for interpretation of function, both in vitro as well as in relevant settings in nature. It is important to assess if native expression levels of non-cognate cross-phyla immunity (e.g. Rothia and Prevotella) protect similarly as the endogenously produced cognate immunity. This experiment could be performed in several ways, for example by deleting the RdnE-I pair and complementing back the Rothia or Prevotella RdnI at the same chromosomal locus, then performing the swarm assay. Alternatively, if there are inducible expression systems available for Proteus, examination of protection under varying levels of immunity induction could be an alternate way to address this question. Western blot analysis comparing cognate to non-cognate immunity protein levels expressed in Proteus could also be important. If the authors were interested in deriving physical binding constants between E and various cognate and non-cognate I (e.g. through isothermal titration calorimetry) that would be a strong set of data to support the claims made. The co-IP data presented in supplemental Figure 6 are nice but are from E. coli cells overexpressing each protein and do not fully address the question of in vivo (in Proteus) native expression.

P. mirabilis strain ATCC29906 does not encode the rdnE and rdnI genes on the chromosome (NCBI BioSample: SAMN00001486) (line 151). Production of the RdnI proteins, including the cognate Proteus RdnI, comes from equivalent transgenic expression vectors. Specifically, the rdnI genes were expressed under the flaA promoter in P. mirabilis strain ATCC29906 (Table 1) for the swarm competition assays found in Figure 2C and Figure 4. This promoter results in constitutive expression in swarming cells (Belas et al., 1991; Jansen et al., 2003). In the revised manuscript, figure 4 Supplement Figure 2 shows the relative RdnI protein levels in these strains; we also clarified the expression constructs in the text (see reviewer 3, comment 1).

Lines 321-324, the authors infer differences between E and I in terms of read recruitment (greater abundance of I) to indicate the presence of orphan immunity genes in metagenomic samples (Figure 5A-D). It seems equally or perhaps more likely that there is substantial sequence divergence in E compared to the reference sequence. In fact, metagenomes analyzed were required only to have "half of the bases on reference E-I sequence receiving coverage". Variation in coverage again could reflect divergent sequence dipping below 90% identity cutoff. I recommend performing metagenomic assemblies on these samples to assess and curate the E-I sequences present in each sample and then recalculating coverage based on the exact inferred sequences from each sample.

This comment raises the challenges with metagenomic analyses. It was difficult to balance specificity to a particular species’ DNA sequence with the prevalence of any homologous sequence in the sample. Given the distinction in binding interactions among the examined four species, we opted to prioritize specificity, accepting that we were losing access to some rdnE and rdnI sequences in that decision. We chose a 90% identity cutoff, which, through several in silica controls, ensured that each sequence we identified was the rdnE or rdnI gene from that specific species. For the Version of Record, we have included analysis with a 70% cutoff in the supplemental information to try to account for sequence divergence by lowering the identity cutoffs as suggested. The data from the 70% identity cutoff was consistent with the original data from the 90% identity cutoff.

A description of gene-level read recruitment in the methods section relating to metagenomic analysis is lacking and should be provided.

Noted. We included the raw code and sequences on the OSF website associated with this manuscript https://osf.io/scb7z/.

Reviewer #3 (Public Review):

Summary:<br /> The authors discovered that the RdnE effector possesses DNase activity, and in competition, P. mirabilis having RdnE outcompetes the null strain. Additionally, they presented evidence that the RdnI immunity protein binds to RdnE, suppressing its toxicity. Interestingly, the authors demonstrated that the RdnI homolog from a different phylum (i.e., Actinomycetota) provides cross-species protection against RdnE injected from P. mirabilis, despite the limited identity between the immunity sequences. Finally, using metagenomic data from human-associated microbiomes, the authors provided bioinformatic evidence that the rdnE/rdnI gene pair is widespread and present in individual microbiomes. Overall, the discovery of broad protection by non-cognate immunity is intriguing, although not necessarily surprising in retrospect, considering the prolonged period during which Earth was a microbial battlefield/paradise.

Strengths:<br /> The authors presented a strong rationale in the manuscript and characterized the molecular mechanism of the RdnE effector both in vitro and in the heterologous expression model. The utilization of the bacterial two-hybrid system, along with the competition assays, to study the protective action of RdnI immunity is informative. Furthermore, the authors conducted bioinformatic analyses throughout the manuscript, examining the primary sequence, predicted structural, and metagenomic levels, which significantly underscore the significance and importance of the EI pair. 

Weaknesses:<br /> (1) The interaction between RdnI and RdnE appears to be complex and requires further investigation. The manuscript's data does not conclusively explain how RdnI provides a "promiscuous" immunity function, particularly concerning the RdnI mutant/chimera derivatives. The lack of protection observed in these cases might be attributed to other factors, such as a decrease in protein expression levels or misfolding of the proteins. Additionally, the transient nature of the binding interaction could be insufficient to offer effective defenses.

Yes, we agree with the reviewer and hope that grant reviewers’ share this colleague’s enthusiasm for understanding the detailed molecular mechanisms of RdnE-RdnI binding across genera. In the revised manuscript, we have continued to emphasize such caveats as the next frontier is clearly understanding the molecular mechanisms for RdnI cognate or non-cognate protection. In the revised manuscript, figure 4 Supplement Figure 2 shows the RdnI protein levels; we also clarified the expression constructs in the text (see reviewer 2, comment 2).

(2) The results from the mixed population competition lack quantitative analysis. The swarm competition assays only yield binary outcomes (Yes or No), limiting the ability to obtain more detailed insights from the data.

The mixed swam assay is needed when studying T6SS effectors that are primarily secreted during Proteus’ swarming activity (Saak et al. 2017, Zepeda-Rivera et al. 2018). This limitation is one reason we utilize in vitro, in vivo, and bioinformatic analyses. Though the swarm competition assay yields a binary outcome, we are confident that the observed RdnI protection is due to interaction with a trans-cell RdnE via an active T6SS. By contrast, many manuscripts report co-expression of the EI pair (Yadev et al., 2021, Hespanhol et al., 2022) rather than secreted effectors, as we have achieved in this manuscript.

(3) The discovery of cross-species protection is solely evident in the heterologous expression-competition model. It remains uncertain whether this is an isolated occurrence or a common characteristic of RdnI immunity proteins across various scenarios. Further investigations are necessary to determine the generality of this behavior.

We agree, which is why we submitted this paper as a launching point for further investigations into the generality of non-cognate interactions and their potential impact on community structure.

Comments from Reviewing Editor:<br />  - In addition to the references provided by reviewer#2, the first manuscript to show non-cognate binding of immunity proteins was Russell et al 2012 (PMID: 22607806).<br />  - IdrD was shown to form a subfamily of effectors in this manuscript by Hespanhol et al 2022 PMID: 36226828 that analyzed several T6SS effectors belonging to PDDExK, and it should be cited.

We appreciate that the reviewer and eLife staff pointed out missed citations. We have incorporated these studies and cited them in the revised manuscript.

[1]

The Proteus RdnE in complex with either the Prevotella or Pseudomonas RdnI showed low confidence at the interface (pIDDT ~50-70%); this AI-predicted complex might support the lack of binding seen in the bacterial two-hybrid assay. On the other hand, the Proteus and Rothia RdnI N-terminal regions show higher confidence at the interface with RdnE. Despite this, the C-terminus of the Proteus RdnI shows especially low confidence (pIDDT ~50%) where it might interact near RdnE’s active site (as suggested by reviewer 1). Given this low confidence and the already stated inaccuracies of AI-generated complexes, we would rather wait for crystallization data to inform potential protection mechanisms of RdnI.

Author response image 1.

eLife assessment

This fundamental paper reports a new biosensor to study G protein-coupled receptor activation by the pituitary adenylyl cyclase-activating polypeptide (PACAP) in cell culture, ex vivo (mouse brain slices), and in vivo (zebrafish, mouse). Convincing data are presented that show the new sensor works with high affinity in vitro, while requiring very high (non-physiological) concentrations of exogenous PACAP when applied to intact tissues. The sensor has not yet been used to detect endogenously released PACAP, raising questions about whether the sensor can be used for its intended purpose. While further work must be pursued to achieve broad in vivo applications under physiological conditions, the new tool will be of interest to cell biologists, especially those studying the large and significant GPCR family.

Joint Public Review:

The manuscript "Engineering of PAClight1P78A: A High-Performance Class-B1 GPCR-Based Sensor for PACAP1-38" by Cola et al. presents the development of a novel genetically encoded sensor, PAClight1P78A, based on the human PAC1 receptor. The authors provide a thorough in vitro and in vivo characterization of this sensor, demonstrating its potential utility across various applications in life sciences, including drug development and basic research.

The main criticism of this manuscript after initial review is that the PACLight1 sensor has not been shown to detect the release of endogenous PACAP, whether in culture, in vivo, or ex vivo. The authors appear to be cognizant of this significant limitation (for a PACAP sensor) but no significant changes to address this limitation are provided in the revision.

While the sensor that is described here is new and the experimental results support the conclusions, the sensor reported here is not suited for the detection of endogenous PACAP release in vivo. In some respects, this manuscript could be seen as a stepping stone for further development either by the authors or other groups. Indeed, in many cases initial versions of genetically encoded sensors undergo substantial development post-publication, as exemplified by the evolution of GCaMP. However, the situation with the PAClight sensor reported here requires a different approach. Unlike GCaMP, which was one of the first genetically encoded calcium indicators, PAClight is another variant in a series of GPCR-fluorophore conjugates, following methodologies similar to those developed in the Lin Tian lab and the multiple GRAB-based sensors from Yulong Li's lab. These sensors have already demonstrated in vivo applicability, setting a standard that PAClight must meet or exceed to confirm its value and novelty.

Given that the title of the manuscript, "Probing PAC1 receptor activation across species with an engineered sensor," implies broader applicability, it potentially misleads readers about the sensor's utility in vivo, where "in vivo" should be understood as referring to the detection of endogenous PACAP release.

To align the manuscript with the expectations set by its title, it is crucial that the authors either provide substantial in vivo validation (ability to detect endogenous release of PACAP) or revise the title and the text to clarify that the sensor is primarily intended to detect exogenously applied PACAP. This clarification will ensure that the manuscript accurately reflects the sensor's current capabilities and scope of use.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Summary:

The manuscript "Engineering of PAClight1P78A: A High-Performance Class-B1 GPCR-Based Sensor for PACAP1-38" by Cola et al. presents the development of a novel genetically encoded sensor, PAClight1P78A, based on the human PAC1 receptor. The authors provide a thorough in vitro and in vivo characterization of this sensor, demonstrating its potential utility across various applications in life sciences, including drug development and basic research.

The diverse methods to validate PAClight1P78A demonstrate a comprehensive approach to sensor engineering by combining biochemical characterization with in vivo studies in rodent brains and zebrafish. This establishes the sensor's biophysical properties (e.g., sensitivity, specificity, kinetics, and spectral properties) and demonstrates its functionality in physiologically relevant settings. Importantly, the inclusion of control sensors and the testing of potential intracellular downstream effects such as G-protein activation underscore a careful consideration of specificity and biological impact.

Strengths:

The fundamental development of PAClight1P78A addresses a significant gap in sensors for Class-B1 GPCRs. The iterative design process -starting from PAClight0.1 to the final PAClight1P78A variant - demonstrates compelling optimization. The innovative engineering results in a sensor with a high apparent dynamic range and excellent ligand selectivity, representing a significant advancement in the field. The rigorous in vitro characterization, including dynamic range, ligand specificity, and activation kinetics, provides a critical understanding of the sensor's utility. Including in vivo experiments in mice and zebrafish larvae demonstrates the sensor's applicability in complex biological systems.

Weaknesses:

The manuscript shows that the sensor fundamentally works in vivo, albeit in a limited capacity. The titration curves show sensitivity in the nmol range at which endogenous detection might be possible. However, perhaps the sensor is not sensitive enough or there are not any known robust paradigms for PACAP release. A more detailed discussion of the sensors's limitations, particularly regarding in vivo applications and the potential for detecting endogenous PACAP release, would be helpful.

We thank the reviewer for carefully analyzing our in vivo data and highlighting the limitation of our results regarding the sensor’s applicability in detecting endogenous PACAP. We added several sections conversing future possibilities for optimization in the discussion (see paragraphs 2-4). We agree that a more specific discussion of the limitations of our study is an important addition to help design future experiments. 

There are several experiments with an n=1 and other low single-digit numbers. I assume that refers to biological replicates such as mice or culture wells, but it is not well defined. n=1 in experimental contexts, particularly in Figure 1, raises significant concerns about the exact dynamic range of the sensor, data reproducibility, and the robustness of conclusions drawn from these experiments. Also, ROI for cell cultures, like in Figure 1, is not well defined. The methods mentioned ROIs were manually selected, which appears very selective, and the values in Figure 1c become unnecessarily questionable. The lack of definition for "ROI" is confusing. Do ROIs refer to cells, specific locations on the cell membrane, or groups of cells? It would be best if the authors could use unbiased methods for image analysis that include the majority of responsive areas or an explanation of why certain ROIs are included or excluded.

We thank the reviewer for the helpful suggestions. We have increased the number of replicates to n=3 for both HEK293T and neuron data depicted in Fig.1c. Furthermore, we have added Fig.1c’ containing the quantification of the maximum responses obtained in the dataset shown in Fig.1c also depicting the single values for each replicate. To clarify the definition of an ROI in our manuscript, we have detailed the process of ROI selection in the Methods section “Cell culture, imaging and quantification section”. Additionally, we also increased mouse numbers for in vivo PACAP infusions in mice (see Figure 4g).

Reviewer #2 (Public Review):

Summary:

The PAClight1 sensor was developed using an approach successful for the development of other fluorescence-based GPCR sensors, which is the complete replacement of the third intracellular loop of the receptor with a circularly-permuted green fluorescent protein. When expressed in HEK cells, this sensor showed good expression and a weak but measurable response to the extracellular presence of PACAP1-38 (a

F/Fo of 43%). Additional mutation near the site of insertion of the linearized GPF, at the C-terminus of the receptor, and within the second intracellular loop produced a final optimized sensor with F/Fo of >1000%. Finally, screening of mutational libraries that also included alterations in the extracellular ligand-binding domain of the receptor yielded a molecule, PAClight1P78A, that exhibited a high ligand-dependent fluorescence response combined with a high differential sensitivity to PACAP (EC50 30 nM based on cytometric sorting of stably transfected HEK293 cells) compared to its congener VIP, (with which PACAP shares two highly related receptors, VPAC1 and VPAC2) as well as several unrelated neuropeptides, and significantly slowed activation kinetics by PACAP in the presence of a 10-fold molar excess of the PAC1 antagonist PACAP6-38. A structurally highly similar control construct, PAClight1P78Actl, showed correspondingly similar basal expression in HEK293 cells, but no PACAP-dependent enhancement in fluorescent properties.

PAClight1P78A was expressed in neurons of the mouse cortex via AAV9.hSyn-mediated gene transduction. Slices taken from PAClight1P78A-transfected cortex, but not slices taken from PAClight1P78Actl-transfected cortex exhibited prompt and persistent elevation of F/Fo after 2 minutes of perfusion with PACAP1-38 which persisted for up to 14 minutes and was statistically significant after perfusion with 3000, but not 300 or 30 nM, of peptide. Likewise, microinfusion of 200 nL of 300 uM PACAP1-38 into the cortex of optical fiber-implanted freely moving mice elicited a F/Fo (%) of greater than 15, and significantly higher than that elicited by application of similar concentrations of VIP, CRF, or enkephalin, or vehicle alone. In vivo experiments were carried out in zebrafish larvae by the introduction of PAClight1P78A into single-cell stage Danio rerio embryos using a Tol2 transposase-based plasmid with a UAS promoter via injection (of plasmid and transposase mRNA), and sorting of post-fertilization embryos using a marker for transgenesis carried in the UAS :

PAClight1P78A construct. Expression of PAClight1P78A was directed to cells in the olfactory bulb which express the fish paralog of the human PAC1 receptor by using the Tg(GnRH3:gal4ff) line, and fluorescent signals were elicited by intracerebroventricular administration of PACAP1-38 at a single concentration (1 mM), which were specific to PACAP and to the presence of PAClight1P78A per se, as controlled by parallel experiments in which PAClight1P78Actl instead of PAClight1P78A was contained in the transgenic plasmid.

Major strengths and weaknesses of the methods and results

The report represents a rigorous demonstration of the elicitation of fluorescent signals upon pharmacological exposure to PACAP in nervous system tissue expressing PAClight1P78A in both mammals (mice) and fish (zebrafish larvae). Figure 4d shows a change in GFP fluorescence activation by PACAP occurring several seconds after the cessation of PACAP perfusion over a two-minute period, and its persistence for several minutes following. One wonders if one is apprehending the graphical presentation of the data incorrectly, or if the activation of fluorescence efficiency by ligand presentation is irreversible in this context, in which case the utility of the probe as a real-time indicator, in vivo, of released peptide might be diminished.

We thank the reviewer for their careful consideration of our manuscript and agree that the activation of PAClight persisting for several minutes at micromolar concentrations could be a potential limitation for in vivo applications. We added a possible explanation for the persisting sensor activation in response to artificial application of PACAP38 in paragraph 3 of the discussion. We agree that this addition eases the interpretation of PAClight signals detected in vivo. 

Appraisal of achievement of aims, and data support of conclusions:

Small cavils with controls are omitted for clarity; the larger issue of appraisal of results based on the scope of the designed experiments is discussed in the section below. An interesting question related to the time dependence of the PACAP-elicited activation of PAClight1P87A is its onset and reversibility, and additional data related to this would be welcome.

We agree that the reversibility of the sensor’s fluorescence is indeed an important feature especially for detecting endogenous PACAP release. Our data indicate that the sensor’s fluorescence is reversible when detecting small to medium doses of PACAP38 (see Figure 4d – Application of 30-300nM) that are presumably closer to physiological concentrations than the non-reversible concentration of 3000nM. Please, see also our new discussion on peptide concentrations in paragraph 4 of our discussion. For future experiments, it is indeed advisable to adjust the interval of repeated applications to the decay of the response at the respective concentration. Considering, the long-lasting downstream effects of endogenous signaling, longer intervals between ligand applications are generally preferred to match more closely the physiological range in which endogenous PAC1 is most likely affective. 

Discussion of the impact of the work, and utility of the methods and data:

Increasingly, neurotransmitter function may be observed in vivo, rather than by inferring in vivo function from in vitro, in cellular, or ex vivo experimentation. This very valuable report discloses the invention of a genetically encoded sensor for the class B1 GPCR PAC1. PAC1 is the major receptor for the neuropeptide PACAP, which in turn is a major neurotransmitter involved in brain response to psychogenic stress, or threat, in vertebrates as diverse as mammals and fishes. If this sensor possesses the sensitivity to detect endogenously released PACAP in vivo it will indeed be an impactful tool for understanding PACAP neurotransmission (and indeed PACAP action in general, in immune and endocrine compartments as well) in future experiments.

However, the sensor has not yet been used to detect endogenously released PACAP. Until this has been done, one cannot answer the question as to whether the levels of exogenously perfused/administered PACAP used here merely to calibrate the sensor's sensitivity are indeed unphysiologically high. If endogenous PACAP levels don't get that high, then the sensor will not be useful for its intended purpose. The authors should address this issue and allude to what kind of experiments would need to be done in order to detect endogenous PACAP release in living tissue in intact animals. The authors could comment upon the success of other GPCR sensors that have been used to observe endogenous ligand release, and where along the pathway to becoming a truly useful reagent this particular sensor is.

We thank the reviewer for highlighting the lack in clarity that the scope of this paper was not intended to cover the detection of endogenous PACAP release. We therefore expanded our discussion to encompass the intended purpose of detecting artificially infused or applied PAC1 agonists, such as conducting fundamental tests of drug specificity and developing new pharmacological ligands to selectively target PAC1. This includes a more detailed discussion of our in vivo findings and a clearer phrasing that stresses the potential application for applied drugs and not endogenous PACAP (see last paragraph in the discussion).

We also agree that little is known about endogenous concentrations of PACAP in the brain. However, we have supplemented our discussion with several references estimating lower concentrations of PACAP and other peptides in vivo, suggesting average PACAP levels below the detection threshold of the sensor. Importantly, within certain brain regions and in closer proximity to release sites, significantly higher concentrations might be reached. Additionally, our data indicate that the concentrations observed under our current conditions do not saturate the sensor in vivo.  

We therefore acknowledge the reviewer’s comment on the sensor’s potential limitations under our current experimental conditions. Hence, we expanded our discussion and suggest the use of higher resolution imaging to potentially reveal loci of high PACAP concentrations, which should be validated by future studies (see also our added discussion in paragraph 4). 

Reviewer #3 (Public Review):

Summary:

The manuscript introduces PAClight1P78A, a novel genetically encoded sensor designed to facilitate the study of class-B1 G protein-coupled receptors (GPCRs), focusing on the human PAC1 receptor. Addressing the significant challenge of investigating these clinically relevant drug targets, the sensor demonstrates a high dynamic range, excellent ligand selectivity, and rapid activation kinetics. It is validated across a variety of experimental contexts including in vitro, ex vivo, and in vivo models in mice and zebrafish, showcasing its utility for high-throughput screening, basic research, and drug development efforts related to GPCR dynamics and pharmacology.

Strengths:

The innovative design of PAClight1P78A successfully bridges a crucial gap in GPCR research by enabling realtime monitoring of receptor activation with high specificity and sensitivity. The extensive validation across multiple models emphasizes the sensor's reliability and versatility, promising significant contributions to both the scientific understanding of GPCR mechanisms and the development of novel therapeutics. Furthermore, by providing the research community with detailed methodologies and access to the necessary viral vectors and plasmids, the authors ensure the sensor's broad applicability and ease of adoption for a wide range of studies focused on GPCR biology and drug targeting.

Weaknesses

To further strengthen the manuscript and validate the efficacy of PAClight1P78A as a selective PACAP sensor, it is crucial to demonstrate the sensor's ability to detect endogenous PACAP release in vivo under physiological conditions. While the current data from artificial PACAP application in mouse brain slices and microinfusion in behaving mice provide foundational insights into the sensor's functionality, these approaches predominantly simulate conditions with potentially higher concentrations of PACAP than naturally occurring levels.

We thank the reviewer for their valuable comments and agree that the use of PAClight for detecting endogenous PACAP will be of big interest for the scientific community and should be a goal for future research. Considering the time, equipment and additional animal licenses necessary, we are convinced that these questions would go beyond the scope of the current paper and might rather be addressed in a follow-up publication. We therefore rephrased the discussion and added more details to clarify further the intended purpose of the current study. Additionally, we added a paragraph in the discussion suggesting experiments needed to validate PAClight for putative future in vivo applications. 

Although the sensor's specificity for the PAC1 receptor and its primary ligand is a pivotal achievement, exploring its potential application to other GPCRs within the class-B1 family or broader categories could enhance the manuscript's impact, suggesting ways to adapt this technology for a wider array of receptor studies. Additionally, while the sensor's performance is convincingly demonstrated in short-term experiments, insights into its long-term stability and reusability in more prolonged or repeated measures scenarios would be valuable for researchers interested in chronic studies or longitudinal behavioral analyses. Addressing these aspects could broaden the understanding of the sensor's practical utility over extended research timelines.

We extend our gratitude to the reviewer for diligently assessing our results. 

Indeed, the very high level of sensitivity that we could achieve in PAClight leads us to think that potentially a grafting-based approach, such as the one we’ve recently described for class-A GPCR-based sensors (PMID: 37474807) could also work for the direct generation of multiple class-B1 sensors based on the optimized fluorescent protein module present in PAClight. Unfortunately, considering the amount of work that testing this hypothesis would entail, we are not able to perform these experiments in the context of this revision, and would rather pursue them as a future project. Nevertheless, we have expanded the discussion of the manuscript with a paragraph with these considerations.

While we lack comprehensive data on the long-term stability of the sensor, our preliminary findings from photometry recordings optimization indicate consistent baseline expression of PAClight and PACLight ctrl over several weeks. Conducting experiments to systematically assess stability would require several months, which is currently impractical due to limitations in tools and licenses for repeated in vivo infusions. Hence, we intend to include these experiments in potential follow-up studies.

Furthermore, the current in vivo experiments involving microinfusion of PACAP near sensor-expressing areas in behaving mice are based on a relatively small sample size (n=2), which might limit the generalizability of the findings. Increasing the number of subjects in these experimental groups would enhance the statistical power of the results and provide a more robust assessment of the sensor's in vivo functionality. Expanding the sample size will not only validate the findings but also address potential variability within the population, thereby reinforcing the conclusions drawn from these crucial experiments.

We agree with the reviewer that a sample size of N=2 is not sufficient for in vivo recordings. We therefore increased the sample size and now present recordings with 5 PAClight1P78A and 4 PACLight-control mice. Of note, the new data validate our previous findings and conclusions and give a better idea of the variability in vivo that we now discuss in much more detail in the discussion (see paragraph 2). 

Reviewer #1 (Recommendations For The Authors):

The lower potency of maxadilan activation might reflect broader implications for ligand-receptor dynamics. Perhaps the authors could discuss the maxadilan binding from a structural perspective, including AlphaFold models. Also, discussing how these findings might influence sensor application in diverse biological contexts would be insightful. Clear definitions and consistent use of these terms are crucial for ensuring that readers understand the methods and results.

We would like to thank the reviewer for the comments. As part of this work, we did not obtain a dose-response curve for maxadilan peptide, and only reported the maximal response of the sensor to a high concentration of the peptide (10 µM). Thus, our findings would rather inform us on the maximal efficacy of the peptide, as opposed to its potency towards the PAC1R. Furthermore, we would like to point out that due to the lack of structural details for any GPCR-based sensor published to date, we cannot make any molecularly accurate conclusion regarding the precise reasons why a different ligand (in this case the sandfly maxadilan) induces a lower maximal efficacy of the response compared to the endogenous cognate ligand of the receptor. We do not believe that AlphaFold models can accurately replace structural information in this regard, especially given the consideration that the aminoacid linker regions between the GPCR and the fluorescent protein, which are a critical determinant of allosteric chromophore modulation by ligand-induced conformational changes, typically obtain the lowest confidence score in all AlphaFold predicted structural models of GPCR-based sensors. Finally, we would like to refer the reviewer to a very nice recent publication (PMID: 32047270) which resolved the structures of each of these peptides bound to the PAC1 receptor-Gs protein complex, which provides accurate molecular details on the different modalities of receptor binding and activation by PACAP138  versus maxadilan.

Reviewer #2 (Recommendations For The Authors):

The authors are congratulated on the meticulous achievement of their aim, i.e. a fluorescence-based sensor for the detection of PACAP with in vivo utility. Whether or not this sensor will have the requisite sensitivity to detect the release of endogenous PACAP within various regions of the nervous system, in response to specific environmental stimuli or changes in brain or physiological state, remains to be determined.

We thank the reviewer for the very positive evaluation of our manuscript and for the suggested additions that will improve the strength of our arguments.

We agree that the in vivo detection of endogenous PACAP will be an important objective for future studies. Due to time, resource and animal license constraints, we are not able to address this objective in our current study, but we now detail possible future experiments in the discussion section. Please see also our answer to the suggested discussion points previously.

Reviewer #3 (Recommendations For The Authors):

To comprehensively assess the sensor's sensitivity and specificity to endogenous PACAP, I recommend conducting additional in vivo experiments where PAClight1P78A is expressed in neurons that endogenously express the Pac1r receptor (using Adcyap1r1-Cre mouse line). These experiments should involve applying sensory or emotional stimuli known to evoke PACAP release or activating upstream PACAP-expressing neurons. Such studies would offer valuable data on the sensor's performance under natural physiological conditions and its potential utility for exploring PACAP's roles in vivo.

We express our gratitude to the reviewer for providing detailed methodological approaches to examine endogenous PACAP release. These suggestions will prove invaluable for future investigations and are important additions to a follow-up publication. As mentioned earlier, we have incorporated some of these approaches into our discussion. Additionally, we have underscored the existing limitations in detecting endogenous PACAP in vivo and emphasized the relevance of PAClight for drug development purposes.

eLife assessment

In this fundamental study, the authors use innovative fine-scale motion capture technologies to study visual vigilance with high-acuity vision, to estimate the visual fixation of free-feeding pigeons. The authors present compelling evidence for use of the fovea to inspect predator cues, the behavioral state influencing the latency for fovea use, and the use of the fovea decreasing the latency to escape of both the focal individual and other flock members. The work will be of broad interest to behavioral ecologists.

Public Review:

The authors used an innovative technic to study the visual vigilance based on high-acuity vision, the fovea. Combining motion-capture features and visual space around the head, the authors were able to estimate the visual fixation of free-feeding pigeon at any moment. Simulating predator attacks on screens, they showed that 1) pigeons used their fovea to inspect predators cues, 2) the behavioural state (feeding or head-up) influenced the latency to use the fovea and 3) the use of the fovea decrease the latency to escape of both the individual that foveate the predators cues but also the other flock members.

The paper is very interesting, and combines innovative technic well adapted to study the importance of high-acuity vision for spotting a predator, but also of improving the behavioural response (escaping). The results are strong and the models used are well-adapted. This paper is a major contribution to our understanding of the use of visual adaptation in a foraging context when at risk. This is also a major contribution to the understanding of individual interaction in a flock.

Author response:

The following is the authors’ response to the original reviews.

eLife assessment

In this fundamental study, the authors use innovative fine-scale motion capture technologies to study visual vigilance with high-acuity vision, to estimate the visual fixation of free-feeding pigeons. The authors present convincing evidence for use of the fovea to inspect predator cues, the behavioral state influencing the latency for fovea use, and the use of the fovea decreasing the latency to escape of both the focal individual and other flock members. The work will be of broad interest to behavioral ecologists.

We thank the editor for his interest and feedback on the manuscript. We hereafter addressed the comments of the reviewer.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The authors were using an innovative technic to study the visual vigilance based on high-acuity vision, the fovea. Combining motion-capture features and visual space around the head, the authors were able to estimate the visual fixation of free-feeding pigeon at any moment. Simulating predator attacks on screens, they showed that 1) pigeons used their fovea to inspect predators cues, 2) the behavioural state (feeding or head-up) influenced the latency to use the fovea and 3) the use of the fovea decrease the latency to escape of both the individual that foveate the predators cues but also the other flock members.

Strengths:

The paper is very interesting, and combines innovative technic well adapted to study the importance of high-acuity vision for spotting a predator, but also of improving the behavioural response (escaping). The results are strong and the models used are well-adapted. This paper is a major contribution to our understanding of the use of visual adaptation in a foraging context when at risk. This is also a major contribution to the understanding of individual interaction in a flock.

Weaknesses:

I have identified only two weaknesses:

(1) The authors often mixed the methods and the results, Which reduces the readability and fluidity of the manuscript. I would recommend the authors to re-structure the manuscript.<br /> (2) In some parts, the authors stated that they reconstructed the visual field of the pigeon, which is not true. They identified the foveal positions, but not the visual fields, which involve different sectors (binocular, monocular or blind). Similarly, they sometimes mix-up the area centralis and the fovea, which are two different visual adaptations.

Thank you for your positive feedback. We addressed these comments by restructuring the methods and result sections as suggested, and by checking the terminology and specific vocabulary used throughout the manuscript.

Reviewer #1 (Recommendations For The Authors):

First, I would like to say that I really enjoyed the manuscript. This is a great contribution to the field.

Thank you for the positive feedback, we highly appreciate it.

Then, I have some comments that I hope, would help the authors to improve the manuscript.

Major comments :

I would recommend the authors to restructure the methods and the results section. In many parts, the models used are presented in the results section, while this should be presented in the methods section.

Thank you for the suggestion, we now have ensured that the model descriptions are presented in the statistic section of the methods.

To me, the introduction is too long (more than 5 pages). It would be beneficial to reduce it considerably. Furthermore, in the introduction, it misses some information about the visual abilities of your species ((visual acuity, visual field, temporal resolution, contrast sensitivity....).

We agree that the introduction was very long and reduced it by removing the “Methodological issues” as well as strongly reducing the “Experimental rationales” to a minimum. We also added the missing information on the visual abilities of the pigeons in the “Experimental rationales” section (see L135-150). Please note, however, that we refer to the temporal resolution of pigeon vision in the method section, to associate it with the information of the used monitor’s resolution.

Minor comments :

Lines 37-39: This needs a reference.

A reference has been added (McFarland, 1977)

Lines 39-41: But see some papers published recently on Harris's hawks.

Thank you for the references, we added the citation as well as a few more papers (Kane et al., 2015; Kano et al., 2018; Miñano et al., 2023; Yorzinski & Platt, 2014).

Lines 41-43: This sentence needs a reference as well.

A reference has been added (Cresswell, 1994; M. H. R. Evans et al., 2018; Inglis & Lazarus, 1981)

Lines 56-103: In this paragraph, head down and head up also depends from the retinal map of the birds! Some birds have visual streak that allow them to see a potential threats while foraging. Please add more information about the importance of photoreceptors distribution.

Thank you for pointing out this issue. We rewrote the sentence L65-69 as follows to include the importance retinal structures.

“In several species, especially those with a broad visual field and specific retinal structures such as the visual streaks, individuals can simultaneously engage in foraging activities while remaining vigilant (Fernández-Juricic, 2012), likely using peripheral vision to detect approaching threats (Bednekoff & Lima, 2005; Cresswell et al., 2003; Kaby & Lind, 2003; Lima & Bednekoff, 1999).”

Lines 76-79: you wrote : ".... favor alternative hypotheses based on their findings". Which findings? You need to explain.

We rewrote this part as follows (L80-81).

“other studies found evidence for the risk dilution (Beauchamp & Ruxton, 2008) and the edge effect (Inglis & Lazarus, 1981) in their study systems.”

Lines 109-110: It would be good to have a representation of what is an area and a fovea, and how it is placed in the eye, what type of fovea exists and how it is related to visual field. Where does it project?

We now give a better description of the pigeon’s visual field in the experimental rationales section that we hope will help the reader understanding the key features of pigeon’s vision (see L135-150). Specifically, we now say in L137-138:

“they have one fovea centrally located in the retina of each eye, with an acuity of 12.6 c/deg (Hodos et al., 1985). Their fovea projects laterally at ~75° into the horizon in their visual field.”

Lines 109-113: You might need to see some new papers here about the fovea. See for instance Bringmann 2019.

Thank you for the suggestion, we now give a more precise definition of the fovea and refer to Bringmann’s paper for more details (L113-114):

“a pit-like area in the retina with high concentration of cone cells where visual acuity is highest, and is responsible for sharp, detailed, and color vision.”

Lines 113-120: Please explain how the visual field is related to fovea? Where is the fovea project in the visual fields?

Similarly to the question above, we now give a more precise description of the pigeon’s visual field (see L135-150).

Line 131-134: For a non-expert, you would need to explain what is micro, meso and macro scale?

These sentences have been removed when shortening the introduction and we are not referring to micro, meso and macro scales anymore.

Lines 134-136: Please explain in one sentence the technique here.

We now explain in one sentence how motion capture enables the tracking of head and body orientation (L130-132):

“Motion capture cameras track with high accuracy the 3D position of markers, which, when attached to the pigeon’s head and body, enables to reconstruct the rotations of the head and body in all directions.”

Line 140: You presented here for the first time the word "foveation". Has this term been used before? If so, please add a reference. If not, please explain what you mean by foveation precisely.

Thank you for noticing this lack. We are now providing the following definition “directing visual focus to the fovea to achieve the clearest vision” in the first place where we mention the term foveation (L149-150).

Lines 146-148: Please explain why this proves that it is appropriate to not record eyes movements, and is this true for every behaviours?

We acknowledge that some small eye movement might occur and reduce the accuracy of the method. This error is considered in the system using the +-10 degrees range around the foveas. The lines the reviewer referred to were removed when shortening the introduction, but we added an explanation in the paragraph describing pigeon vision to make it clearer (L147-150):

“Yet, it should be noted that their eye movement was not tracked in our system, although it is typically confined within a 5 degrees range (Wohlschläger et al., 1993). We thus considered this estimation error of the foveation (directing visual focus to the fovea to achieve the clearest vision) in our analysis, as a part of the error margin (see Methods).”

Lines 161-163: What is the frontal and binocular field for? You would need to explain the different fields of view and what they are supposed to be for.

Furthermore, does the visual field of pigeon have been studied? If so, you would need to add more information about it.

This information is now given in the new paragraph describing the pigeon’s vision in the  “Experimental rationales” section (see L135-150).

Figure 1: It is not clear here which panels correspond to a, b or c. Please use some boxes to clarify it.

Thank you for the comment, we now have made the figure’s sub-panels clearer.

Lines 193-194: You wrote "... such as foveas (also known as the area centralis). No, this is not the same.

(1) In some species, you have two foveas, one placed centrally in the retina, one place temporally. So the fovea is not the area centralis.

(2) Second, some species do have an area centralis but without a fovea.

Thank you for pointing out the inaccuracy. In this case, we were referring specifically to the pigeon’s fovea which is sometimes referred to as “area centralis”, but we now changed the sentence as follow to avoid any confusion (L174-175):

“The initial two hypotheses (Hypotheses 1 and 2) aim to examine whether foveation correlates with predator detection.”

Lines 192-212: I did not understand the logic of the hypotheses numbers? Why do you have 2.1 but not 3.1 for instance? And if you have two hypotheses for the within a global one (for instance, 2.1 and 2.2), what is the main hypothesis 2? You should explain more here because we get lost here and in the result section as well.

We recognize this section might have appeared confusing to the reader. In short, we had four main hypotheses: 1) the fovea is used to evaluate predator cues, 2) the latency to foveate is related to vigilance behaviors. These first 2 hypotheses aimed to determine if the latency to foveate on the predator cue could be related to the detection. 3) foveation is related to the escape response of the pigeons and 4) there is a collective influence in the escape response. We further divided some of the hypotheses into 2 sub-hypotheses whenever 2 different tests were used to answer the same question. We have modified this section to be clearer.

Lines 224-229: Where are the figures and statistics for these results?

These results are presented in Table S1. We apologize for forgetting to add this reference and have now added it (L211).

Lines 229-231: This should be in the method section.

This model explanation (as well as all other hereafter mentioned) have been moved to the method section as suggested.

Lines 248-252: This should be in the method section. Furthermore, you should better explain the model selection.

Please see earlier comment. Additionally, we are now better explaining how the model has been built.

Figure 2: It is not clear on the figure which letters correspond to which panels. Please improve the readability of the figure.

It was modified accordingly.

Lines 274-278: This should be in the method section.

Please see earlier comment.

Line 281: The "Fig.3" should be mentioned in the previous sentence.

It was modified accordingly.

Figure 3: Please explain why the latency to foveate had negative values in Fig.2 but not here, and not in Fig. 4 as well. This again highlights that we missed a number of information in the methods about the transformation of the data and the model selection.

The variable presented in Fig 2d is not the latency to foveate but the “Normalized frequency at which the object was observed within foveal regions” (hypothesis 1). It represents the amount of time the object was lying within one of the foveal regions of the individual (“how long the pigeons foveated on it”), further normalized to unit sum to make all objects comparable. This variable was indeed logit-transformed (hence the negative value) to improve residual fit in the model, but this information (as well as other transformations) are always clearly stated on the axis caption of the graphs. Additionally, we now have improved the statistical analysis section to make the model used for each hypothesis testing clearer. But please let us know if you have suggestions for a further improvement in terms of presentation.

Lines 297-301: This should be in the method section.

Please see earlier comment.

Lines 301-305: Fig. 3 b and c only referred to the two first factors. Please add more figures for the other factors. This could be in supp. Mat.

We added the 3 graphs for the proportion of time foveating on the monitor, the saccade rate and the proportion of time foveating on conspecifics in the supplementary (Fig S6).

Lines 306-309: This should be in methods, and you should have explained in methods how you performed your model selection.....

We prefer leaving this paragraph in the result section, as it was intended to give the reader extra information on the predictive power of the different variables (by comparing the effectiveness of the models including one variable at a time, all the rest being equal) and not on the model selection per se. However, we now explain our goal better in the statistics section regarding this analysis (L635-636):

“We further tested the relative predictive power of the different test variables by comparing the resulting models’ efficiency using AIC scores.”

Lines 317-319: This should be in the method section.

Please see earlier comment.

Lines 320-322: This should be in the method section.

Please see earlier comment.

Lines 332-334: This should be in the method section.

Please see earlier comment.

Lines 334-336: Then, if this is not significant, you cannot say that.

Thank you for noticing the inaccuracy, we have now rephrased it as (L298-299):

“Earlier foveation of the first pigeon was not significantly related to an earlier escape responses among the other flock members, although there was a trend (χ2(1) = 3.66, p = 0.0559).”

Line 336: Please explain why you did different models. We missed a lot of information in the method about your strategy for statistics.?

We have now added a lot more information on the models in the statistics section, according to this comment as well as the previous ones. We hope the explanations of the analyses are now clearer to the reader.

Lines 339-349: This should be in the method section.

Please see earlier comment.

Results section: As you may have understood, there are too many sentence that should be moved into the method section. Futhermore, I would recommend to modify the headdings so that they are more biologically speaking. Similarly to what you have done in the discussion section.

Thank you for the comments. We agree with most of them, and have modified the manuscript accordingly. Additionally, we now use the same headings in the results section as the ones used in the discussion to make the text easier to follow.

Lines 500-501: What were the body weight of the pigeon? At which weight of their full weight they were?

This information is now added (492 ± 41g; mean ± SD). We did not control the amount of food during our experiments and only ensured 24h without food by feeding the pigeons after the experiment was completed. This information was added as follows (L454-456):

“On experimental days, they were fed only after the experiments was completed; this ensures 24-hour no feeding at the time of the experiment, although we did not control the amount of the food over the course of the experimental periods.”

Line 522-523: Those screens are very good for pigeons.

Thank you for the positive comment, we indeed tried to match bird vision as close as possible.

Lines 527-528: At which frequency was produced the moving stimulus? Your screen can display up to 144Hz, which is very good. But can your laptop do it? If not, it is important to mention it as pigeons may have a temporal resolution of vision up to 149Hz.

Our laptop indeed supports 144Hz display. In addition, we now mention the temporal resolution of pigeon vision (L480-482).

“We specifically chose a monitor with high temporal resolution to match the pigeon’s Critical Flicker Fusion Frequency (threshold at which a flickering light is perceived by the eye as steady) that reaches up to 143Hz (Dodt & Wirth, 1954).”

Lines 555-572: Did you use a control shape in your experiment? Indeed, they may escape because of a moving pattern but not a predator shape?

We did not use a control shape, as the aim of the experiment was not to directly test the effect of the shape itself. We designed the predator cue to resemble an approaching predator to ensure a response from the pigeons, but it might be that other shapes would have worked as well.

Lines 588-589: Please explain why the coordinate system of the pigeon's head is considered as the visual field?

From what I have understood, you did not reconstruct the visual fields, but only the position of the fovea. This should be noted like this as visual field involves more than a sphere around the head (binocular and monocular sectors, blind sectors, vertical extension....).

Thank you for noticing the inaccuracy, we indeed did not consider other sectors of the visual field and therefore rephrased it as (L551): “the location of the objects and conspecifics from the pigeon’s perspective”.

Lines 601-604: How much does it represent?

As this was estimated by visual inspection, we do not have the exact percentage of data loss that was caused by grooming. However, because of the number of cameras in the SMART BARN motion capture system, it is reliable in detecting markers inside the space in “ideal” conditions (without occlusion). For example, a similar set-up found marker track loss of only <1% using a model bird (Itahara & Kano 2022)

Itahara, A., & Kano, F. (2022). “Corvid Tracking Studio”: A custom-built motion capture system to track head movements of corvids. Japanese Journal of Animal Psychology, 72(1), 1–16. https://doi.org/10.2502/janip.72.1.1

Lines 610-612: You would need to cite Wood 1917 and Hodos et al. 1991 who described the presence of a fovea in this species.

We added both citations to the manuscript.

Line 611: Again, the fovea is not egal to area centralis.

Thank you, we changed it as well.

Lines 625-626: you wrote "... in a few instances....". Please explain more. How many? What proportion?

This happened in 9 observations out of 120. We now specify it in the text as well (L587-589):

“in a few instances (9 out of 120 observations), pigeons foveated on the model predator after the looming stimulus had disappeared, but these cases were excluded from our analysis.”

Lines 640-653: We missed a lot of information in the section "statistical analysis". If you moved most of the sentence from the results that describe the methods in the method section, that would be much better. Furthermore, you would need to explain more what statistics you used, which model selection, what type of data transformation....

We agree this section lacked information, and we moved the information from the result to the statistics section.

Supplmentary materials: boxplots from Fig. S1 and S2 are too small and impossible to read. Please improve the readability.

We now have enlarged these plots to make them more readable.

А ничего, что после значка окончания материала ещё текст?

А вот тут и везде ниже (а в случае выше нет) в первой строке консоли стоит $, а в коде перед нет. Так надо?

Всё-таки кажется непрозрачным. Заменить что на что -- ошибочное управление (норма что чем), но большинство так говорит. И следовательно, может прочитать. Тогда получится, что заменили ссылку русскоязычным аналогом. Корректно было бы что-то вроде "заменим ссылку в ней ссылкой на русскоязычную страницу".

Это выглядит так, как задумано? Кажется, что предполагалась какая-то таблица или хотя бы какое-то подобие системы, а так приходится соотносить числовые данные с текстовыми, прикладывая некоторые мозговые усилия.

Kepha = Rock "Peter" is an anachronistic(?) translation. The English name does not mean "rock" in the English mind.

На скрине после плюсов пробелы. Это что собой представляет? В зависимости от сути может быть с пробелом или без, не знаю, на что тут опереться.

Оторвано.

А вот чуть ниже 43 -- это вот этот вот размер? Не должны быть одинаковыми?

Оторвано.

У меня в конце строки.

eLife assessment

The authors performed extensive coarse-grained molecular dynamics simulations of 140 different prion-like domain variants to interrogate how specific amino acid substitutions determine the driving forces for phase separation. The analyses are solid, and the predictive scaling laws can aid in identifying potential phase-separating regions in uncharacterized proteins. Overall, this is a valuable contribution to the field of biomolecular condensates. It exemplifies how data-driven methodologies can uncover new insights into complex biological phenomena.

Reviewer #1 (Public Review):

Summary:

In this preprint, the authors systematically and rigorously investigate how specific classes of residue mutations alter the critical temperature as a proxy for the driving forces for phase separation. The work is well executed, the manuscript well-written, and the results reasonable and insightful.

Strengths:

The introductory material does an excellent job of being precise in language and ideas while summarizing the state of the art. The simulation design, execution, and analysis are exceptional and set the standard for these types of large-scale simulation studies. The results, interpretations, and Discussion are largely nuanced, clear, and well-motivated.

Weaknesses:

This is not exactly a weakness, but I think it would future-proof the authors' conclusions to clarify a few key caveats associated with this work. Most notably, given the underlying implementation of the Mpipi model, temperature dependencies for intermolecular interactions driven by solvent effects (e.g., hydrophobic effect and charge-mediated interactions facilitated by desolvation penalties) are not captured. This itself is not a "weakness" per se, but it means I would imagine CERTAIN types of features would not be well-captured; notably, my expectation is that at higher temperatures, proline-rich sequences drive intermolecular interactions, but at lower temperatures, they do not. This is likely also true for the aliphatic residues, although these are found less frequently in IDRs. As such, it may be worth the authors explicitly discussing.

Similarly, prior work has established the importance of an alpha-helical region in TDP-43, as well as the role of aliphatic residues in driving TDP-43's assembly (see Schmidt et al 2019). I recognize the authors have focussed here on a specific set of mutations, so it may be worth (in the Discussion) mentioning [1] what impact, if any, they expect transient or persistent secondary structure to have on their conclusions and [2] how they expect aliphatic residues to contribute. These can and probably should be speculative as opposed to definitive.

Again - these are not raised as weaknesses in terms of this work, but the fact they are not discussed is a minor weakness, and the preprint's use and impact would be improved on such a discussion.

Reviewer #2 (Public Review):

This is an interesting manuscript where a CA-only CG model (Mpipi) was used to examine the critical temperature (Tc) of phase separation of a set of 140 variants of prion-like low complexity domains (PLDs). The key result is that Tc of these PLDs seems to have a linear dependence on substitutions of various sticker and space residues. This is potentially useful for estimating the Tc shift when making novel mutations of a PLD. However, I have strong reservations about the significance of this observation as well as some aspects of the technical detail and writing of the manuscript.

(1) Writing of the manuscript: The manuscript can be significantly shortened with more concise discussions. The current text reads as very wordy in places. It even appears that the authors may be trying a bit too hard to make a big deal out of the observed linear dependence.

The manuscript needs to be toned done to minimize self-promotion throughout the text. Some of the glaring examples include the wording "unprecedented", "our research marks a significant milestone in the field of computational studies of protein phase behavior ..", "Our work explores a new framework to describe, quantitatively, the phase behavior ...", and others.

There is really little need to emphasize the need to manage a large number of simulations for all 140 variants. Yes, some thoughts need to go into designing and managing the jobs and organizing the data, but it is pretty standard in computational studies. For example, large-scale protein ligand-free energy calculations can require one to a few orders of magnitude larger number of runs, and it is pretty routine.

When discussing the agreement with experimental results on Tm, it should be noted that the values of R > 0.93 and RMSD < 14 K are based on only 16 data points. I am not sure that one should refer to this as "extended validation". It is more like a limited validation given the small data size.

Results of linear fitting shown in Eq 4-12 should be summarized in a single table instead of scattering across multiple pages.

The title may also be toned down a bit given the limited significance of the observed linear dependence.

(2) Significance and reliability of Tc: Given the simplicity of Mpipi (a CA-only model that can only describe polymer chain dimension) and the low complexity nature of PLDs, the sequence composition itself is expected to be the key determinant of Tc. This is also reflected in various mean-field theories. It is well known that other factors will contribute, such as patterning (examined in this work as well), residual structures, and conformational preferences in dilute and dense phases. The observed roughly linear dependence is a nice confirmation but really unsurprising by itself. It appears how many of the constructs deviate from the expected linear dependence (e.g., Figure 4A) may be more interesting to explore.

The assumption that all systems investigated here belong to the same universality class as a 3D Ising model and the use of Eqn 20 and 21 to derive Tc is poorly justified. Several papers have discussed this issue, e.g., see Pappu Chem Rev 2023 and others. Muthukumar and coworkers further showed that the scaling of the relevant order parameters, including the conserved order parameter, does not follow the 3D Ising model. More appropriate theoretical models including various mean field theories can be used to derive binodal from their data, such as using Rohit Pappu's FIREBALL toolset. Imposing the physics of the 3D Ising model as done in the current work creates challenges for equivalence relationships that are likely unjustified.

While it has been a common practice to extract Tc when fitting the coexistence densities, it is not a parameter that is directly relevant physiologically. Instead, Csat would be much more relevant to think about if phase separation could occur in cells.

Reviewer #3 (Public Review):

Summary:

"Decoding Phase Separation of Prion-Like Domains through Data-Driven Scaling Laws" by Maristany et al. offers a significant contribution to the understanding of phase separation in prion-like domains (PLDs). The study investigates the phase separation behavior of PLDs, which are intrinsically disordered regions within proteins that have a propensity to undergo liquid-liquid phase separation (LLPS). This phenomenon is crucial in forming biomolecular condensates, which play essential roles in cellular organization and function. The authors employ a data-driven approach to establish predictive scaling laws that describe the phase behavior of these domains.

Strengths:

The study benefits from a robust dataset encompassing a wide range of PLDs, which enhances the generalizability of the findings. The authors' meticulous curation and analysis of this data add to the study's robustness. The scaling laws derived from the data provide predictive insights into the phase behavior of PLDs, which can be useful in the future for the design of synthetic biomolecular condensates.

Weaknesses:

While the data-driven approach is powerful, the study could benefit from more experimental validation. Experimental studies confirming the predictions of the scaling laws would strengthen the conclusions. For example, in Figure 1, the Tc of TDP-43 is below 300 K even though it can undergo LLPS under standard conditions. Figure 2 clearly highlights the quantitative accuracy of the model for hnRNPA1 PLD mutants, but its applicability to other systems such as TDP-43, FUS, TIA1, EWSR1, etc., may be questionable.

The authors may wish to consider checking if the scaling behavior is only observed for Tc or if other experimentally relevant quantities such as Csat also show similar behavior. Additionally, providing more intuitive explanations could make the findings more broadly accessible.

The study focuses on a particular subset of intrinsically disordered regions. While this is necessary for depth, it may limit the applicability of the findings to other types of phase-separating biomolecules. The authors may wish to discuss why this is not a concern. Some statements in the paper may require careful evaluation for general applicability, and I encourage the authors to exercise caution while making general conclusions. For example, "Therefore, our results reveal that it is almost twice more destabilizing to mutate Arg to Lys than to replace Arg with any uncharged, non-aromatic amino acid..." This may not be true if the protein has a lot of negative charges.

I am surprised that a quarter of a million CPU hours are described as staggering in terms of computational requirements.

eLife assessment

This valuable study tests the functional role of food-washing behavior in removing tooth-damaging sand and grit in long-tailed macaques and whether dominance rank predicts the level of investment in the behavior. The evidence that food-washing is deliberate is compelling, but the evidence for variable and adaptive investment depending on rank is incomplete given confounding between sex and rank and limited sample size. A more careful and perhaps restrained interpretation of the findings, as well as a connection to the existing literature on optimal foraging theory, would increase the value of the study to its intended audience, i.e. researchers interested in foraging behavior, cognition, and primate evolution.

Reviewer #1 (Public Review):

Summary:

In this paper, the authors had 2 aims:

(1) Measure macaques' aversion to sand and see if its' removal is intentional, as it is likely in an unpleasurable sensation that causes tooth damage.

(2) Show that or see if monkeys engage in suboptimal behavior by cleaning foods beyond the point of diminishing returns, and see if this was related to individual traits such as sex and rank, and behavioral technique.

They attempted to achieve these aims through a combination of geochemical analysis of sand, field experiments, and comparing predictions to an analytical model.

The authors' conclusions were that they verified a long-standing assumption that monkeys have an aversion to sand as it contains many potentially damaging fine-grained silicates and that removing it via brushing or washing is intentional.

They also concluded that monkeys will clean food for longer than is necessary, i.e. beyond the point of diminishing returns, and that this is rank-dependent.

High and low-ranking monkeys tended not to wash their food, but instead over-brushed it, potentially to minimize handling time and maximize caloric intake, despite the long-term cumulative costs of sand.

This was interpreted through the *disposable soma hypothesis*, where dominants maximize immediate needs to maintain rank and increase reproductive success at the potential expense of long-term health and survival.

Strengths:

The field experiment seemed well-designed, and their quantification of physical and mineral properties of quartz particles (relative to human detection thresholds) seemed good relative to their feret diameter and particle circularity (to a reviewer who is not an expert in sand). The *Rank Determination* and *Measuring Sand* sections were clear.

In achieving Aim 1, the authors validated a commonly interpreted, but unmeasured function, of macaque and primate behavior-- a key study/finding in primate food processing and cultural transmission research.

I commend their approach in developing a quantitative model to generate predictions to compare to empirical data for their second aim.

This is something others should strive for.

I really appreciated the historical context of this paper in the introduction, and found it very enjoyable and easy to read.

I do think that interpreting these results in the context of the *disposable soma hypothesis* and the potential implications in the *paleolithic matters* section about interpreting dental wear in the fossil record are worthwhile.

Weaknesses:

Most of the weaknesses in this paper lie in statistical methods, visualization, and a missing connection to the marginal value theorem and optimal foraging theory.

I think all of these weaknesses are solvable.

The data and code were not submitted. Therefore I was unable to better understand the simulation or to provide useful feedback on the stats, the connection between the two, and its relevance to the broader community.

(1) Statistics:

(a) AIC and outcome distributions

The use of AIC for hierarchical models, and models with different outcome distributions brought up several concerns.

The authors appear to use AIC to help inform which model to use for their primary analyses in Tables S1 and S2. It is unclear which of these models are analyzed in Tables S3 and S4.

AIC should not be used on hierarchical models, and something like WAIC (or DIC which has other caveats) would be more appropriate.

Also, using information criteria on Mixture Models like Negative Binomials (aka Gamma-Poisson) should be done with extreme caution, or not at all, as the values are highly sensitive to the data structure.

Some researchers also say that information criteria should not be used to compare models with different outcome distributions - although this might be slightly less of a concern as all of your models are essentially variations on a Poisson GLM.

Discussion on this can be found in McElreath Statistical Rethinking (Section 12.1.3) and Gelman et al. BDA3 (Chapter 7).

Choosing an outcome distribution, based on your understanding of the data generating process is a better approach than relying on AIC, especially in this context where it can be misleading.

(b) Zeros

I also had some concerns about how zeros were treated in the models.

In lines 217-218, they mentioned that "if a monkey consumed a cucumber slice without brushing or washing it, the zero-second duration was included in both GLMMs."

This zero implies no processing and should not be treated as a length 0 duration of processing.

This suggests to me that a zero-inflated poisson or zero-inflated negative binomial, would be the best choice for modelling the data as it is essentially a 2-step process:<br /> (i) Do they process the cucumber at all?<br /> (ii) If so do they wash or brush, and how is this predicted by rank and treatment?

(2) Absence of Links to Foraging Theory

Optimal cleaning time model: the optimality model was not well described including how it was programmed. Better description and documentation of this model, along with code (Mathematica judging from the plot?) is needed.

There seems to be much conceptual and theoretical overlap with foraging theory models that were not well described - namely the *marginal value theorem (Charnov (1976), Krebs et al. (1974),) and its subsequent advances* (see https://doi.org/10.1016/j.jaa.2016.03.002 and https://doi.org/10.1086/283929 for examples).

In the suggestions, I attached the R code where I replicated their model to show that it is *mathematically identical to the marginal value theorem*. This was not mentioned at all in the text or citations.

This is a well-studied literature since the 1970's and there is a history of studies that compare behavior to an optimality model and fail (or do find) instances where animals conform or diverge with its predictions (https://doi.org/10.1146/annurev.es.15.110184.002515). This link should be highlighted, and interpreting it in that theoretical context will make it more broadly applicable to behavioral ecologists.

The data was subsetted to include instances where there were < 3 monkeys present to avoid confounds of rank, but it is important to know that optimal behavior might vary by individual, and can change in a social context depending on rank (see https://doi.org/10.1016/j.tree.2022.06.010). Discussion of this, and further exploration of it in the data would strengthen the overall contribution of this manuscript to the field, but I understand that the researchers wish to avoid that in this paper for it is a complex topic, which this dataset is uniquely suited to address.

(3) Interpretation and validity of model relative to data

In lines 92-102, they present summary statistics (I think) showing that time spent brushing and washing is consistent with washing or brushing to remove sand.

In the **mitigating tooth wear** section (line 73) and corresponding Figure S1 showing surface sand removed, more detail about how these numbers were acquired, and statistical modelling, is needed.

This is important as uncertainty and measurement error around these metrics are key to the central finding and interpretation of Aim 2 in this paper.

It appears that the researchers simulated the monkey's brushing and washing behaviors (similar to https://doi.org/10.1007/s10071-009-0230-3).

How many researchers simulated monkey behavior and how many times?

What are the repeat points in Figure S1?

What is the number of trials or number of people?

This effect appears stronger for washing than brushing as well - if so, why?

More info about this data, and the uncertainty in this is important, as it is key to the second central claim of this paper.

The estimates of removing between 76% +/- 7 and 93% +/- 4 of sand (visualized in Figure S1), are statistical estimates.

I would find the argument more convincing if after propagating for the uncertainty in handling in sand removal rates, and the corresponding half-saturation constants, if this processing for food is too long, after accounting for diminishing returns held true.<br /> It is very possible that after accounting for uncertainty and variation in handling time and removal rates, the second result may not hold true.

I was not able to convince myself of this via reanalysis as the description of the data in the text was not enough to simulate it myself.

Essentially, this would imply that in Figure 3 the predicted value would have some variation around it (informed by boundary conditions of time being positive, and percents having floors and ceilings) and that a range of predicting cleaning times (optimal give-up times) would be plotted in Figure 3.

This could be accomplished in a Bayesian approach, Or by simply plotting multiple predictions given some confidence interval around, c and h.

Reviewer #2 (Public Review):

Summary:

This field experiment aimed to assess what motivates macaque monkeys to clean food items prior to consumption and the relative costs and benefits of different cleaning approaches (manually brushing sand from food versus dousing food items in water). The experiment teases apart if/how the benefits of these approaches are mediated by the amount of debris on food and the monkeys' rank in terms of the costs of consuming sand versus the time and energy required to remove it. The authors not only examined the behavioral responses of wild macaques to three conditions of food sand contamination but also tested the relative costs of consuming different levels and sizes of sand particulates. Through this, the authors propose considerations of the macaques' motivations to clean food and the balance they take in energetic gains from consuming food versus the costs of cleaning food and consuming sand. Their data reveal that food washing is more effective in removing sand, but more costly than manually brushing off sand. This study also revealed that only mid-ranked monkeys washed their food, while high and low-ranked monkeys were more likely to remove sand via brushing it off food with their hands.

Strengths:

This study provides a very in-depth consideration of the motivations of macaques to clean their food, and the relative costs and benefits of different food cleaning techniques. Not only did the study test the behavior of wild macaques via a simple yet elegant field study, but they also performed a detailed analysis of the sand particulates to understand the level of potential tooth wear that consuming it could result in. By relying on a wild group of macaques that have been part of a long-term study site, the team also had detailed behavioral data on the population to allow for rank assessments of the animals. This comprehensive study provides important foundational information for a better understanding of how and why macaques clean food, that inform existing and future considerations of this as a potential cultural behavior.

Weaknesses:

As currently written, the paper does not provide sufficient background on this population of animals and their prior demonstrations of food-cleaning behavior or other object-handling behaviors (e.g., stone handling). Moreover, the authors' conclusions focus on the behavior of high-ranked animals, but subordinate animals also showed similar behavioral patterns and they should be considered in more detail too.

Reviewer #3 (Public Review):

This paper provides evidence that food washing and brushing in wild long-tailed macaques are deliberate behaviors to remove sand that can damage tooth enamel. The demonstration of the immediate functional importance of these behaviors is nicely done. However, the paper also makes the claim that macaques systematically differ in their investment in food cleaning because of rank-dependent differences in their costs and benefits. This latter conclusion is not, in my view, well-supported, for several reasons.

First, as is typical in many primate studies, the authors construct sex-specific ordinal rank hierarchies. This makes sense since hierarchies for males and hierarchies for females are determined by different processes and have different consequences. However, if I understand it correctly, they are then lumped together in all statistical analyses of rank, which makes the apparent rank effect very difficult to understand. The challenge of interpretation is increased because there are twice as many adult females in the group as adult males, so the rank is confounded by sex (because all low-rank values are adult females).

Second, because only one social group is being studied, the conclusions about rank may be heavily driven by individual identity, not rank per se. An analysis involving replicate social groups (which granted, may be impossible here) or longitudinal data showing a change in behavior following a change in rank would be much more compelling.

Third, there is no evidence presented on the actual fitness-related costs of tooth wear or the benefits of slightly faster food consumption. Support for these arguments is provided based on other papers, some of which come from highly resource-limited populations (and different species). But this is a population that is supplemented by tourists with melons, cucumbers, and pineapples! In the absence of more direct data on fitness costs and benefits, the paper makes overly strong claims about the ability to explain its observations based on "immediate energetic requirements" (abstract), "difference...freighted with fitness consequences" (line 80), and "pressing energetic needs"/"live fast, die young" (lines 121-122--there is no evidence that tooth wear is associated with morbidity or mortality here). The idea that high-ranking animals are "sacrificing their teeth at the altar of high rank" seems extreme.

you are Rock Jesus' spoke Aramaic. Cephas

Giving advice to others can also strengthen your own understanding.

We need to keep an open mind to every piece of feedback so that we can make progress.

This step is critical, as everyone in the writing workshop needs to be knowledgeable on the subject at hand. As if someone is not up to date they can not peer review and help others fix their writing. This step also brings up a great point in that it is disrespectful to do this in a writing workshop. As you are not helping anyone in this case.

cloud run cold start

https://www.derstandard.at/story/3000000228970/fluglinien-muessen-co2-vebrauch-drastisch-reduzieren-doch-es-fehlt-an-gruenem-sprit

acknowledge but does not accept

God did not condemn him or call him foolish -> not a rebuke or punishment but Childe suggests affection, tenderness, intimacy and acknowledging the kinship/bond

Sense of a child being simple-minded and ignorant

undramatic response -> very simple, direct and unostentatious

if one stops himself from serving his own need, they deserve the burden they get

if you do not act to serve your own interests

repetition of the opening line

suggestion that this is what God is holding over them -> that one shouldnt care or fear about God and judgement and punishment -> nothing more than a symbol/emblem

says that rejecting God's law is a sort of revelation to have

refers to reason and rationality but he describes it as pettie, while law is a rope of sand

inconsequential and easy to do away with

they are the ones who think that the rope of sand is actually strong and will not easily disintegrate like sand

no reward - concept of abundance

uses very perjorative terms to describe the concept of morality

like the collar, this refers to right and wrong

now irrelevant and no longer fresh

constantly wondering and debating and conflicting with onself -> cold dispute as if its no longer warm (irrelevant, old, has gone on for a long time)

once again signify restraint and confinement

recover what he is wasted

pleasures ->

the years that he lost

sigh -> complaint and blown as if he has wasted it -> the 'age' that he has spent away

No earthly recognition or reward and his solution is to abandon and forsake

Suggests restraint or a controlling authority -> lack of freedom and externally imposed control

Also worn by ministers and priests -> could be about rebelling

Collar is a pun on choleric -> anger and situation of rage

being abroad

metaphor of harvest with corn -> symbolise the harvest collectively and just rewards on earth (worldly rewards)

Throughout the poem, shortness of the lines are used for emphatic effect -> when he makes a declaration

he will not even get one small/sweet reward

he does not get what he toils for but he also cannot restore what he has sacrificed -> second metaphor is more limited

to ask for authority and sue for justice -> shall i be in the condition of begging/being a supplicant

always -> constant suffering

desiring and wanting something else -> pine suggests suffering for want of something else and an unfulfilled longing

christians are restrained by a sense of morality and the bible -> to perceive it as a collar, you are already in a state of defiance/resentment

Very inspiring thought related to power and word.

Version 4 of this preprint has been peer-reviewed and recommended by Peer Community in Mathematical and Computational Biology.<br /> See the peer reviews and the recommendation.

It seems like you can see something too much and become desensitized to it, or not exposed enough and lack understanding on a topic. So ideally, we have a medium amount of bad experiences to relate to others?? Also the pdf for writ 340 wasn't working so I'm doing this here

I think that generally, one can relate to and put yourself in the same feelings even if you don't know the same experience that made your friend sad?

Que the Adventure Time empathy song: https://www.google.com/search?q=empathy+empathy+adventure+time&oq=empathy+empathy&gs_lcrp=EgZjaHJvbWUqBwgAEAAYgAQyBwgAEAAYgAQyCQgBEEUYORiABDIHCAIQABiABDIHCAMQABiABDIICAQQABgWGB4yBggFEEUYPDIGCAYQRRg9MgYIBxBFGD3SAQgzMTQyajBqN6gCALACAA&sourceid=chrome&ie=UTF-8#fpstate=ive&vld=cid:3a147137,vid:Wa9C7omTKOo,st:0

I've always heard it as: sympathy is feeling FOR someone, empathy is feeling WITH someone

https://www.chicagotribune.com/1988/09/11/playing-with-ire/

This is fascinating. - Ask Ashley

La subjetividad y la objetividad están interrelacionadas en la investigación social, ya que la perspectiva subjetiva de los individuos enriquece el análisis de los fenómenos sociales, ofreciendo una comprensión más completa.

Aquí Gonzalez apela a una reflexión de Viktor Frankl, quien destaca cómo, incluso en las situaciones más extremas, las personas buscan un propósito y un sentido en sus vidas. Esto apoya la idea de que nuestra forma de entender y darle sentido a nuestras experiencias (subjetividad) es una parte importante de la investigación social, y no se puede ignorar al estudiar fenómenos humanos.

Respondiendo a la pregunta ¿Qué aspectos determinan la objetividad y la subjetividad en la investigación de fenómenos sociales? Fernando González sostiene que la objetividad y la subjetividad no son opuestas sino complementarias en la investigación social. Mientras que la objetividad tradicional puede simplificar y limitar la comprensión de fenómenos complejos a datos cuantificables, la subjetividad permite explorar la profundidad y riqueza de las experiencias humanas y contextos culturales. En lugar de reducir la complejidad a simples categorías, integrar la subjetividad en la investigación ofrece una perspectiva más creativa, lo cual es necesario para desafiar sistemas autoritarios y de control que imponen visiones más conservadoras

Aquí Fernando González argumenta a través de Foucault que, en lugar de simplemente etiquetar lo que no entendemos como una enfermedad mental, deberíamos tratar de comprender el contexto y la perspectiva del individuo. Esto subraya que nuestra manera de interpretar y entender el comportamiento de las personas, basada en sus propias experiencias y sentimientos, es crucial para la investigación social.

Esta mención de Giordano Bruno refuerza la idea de que nuestra propia perspectiva es parte importante del conocimiento que generamos, demostrando que la forma en que entendemos y analizamos el mundo está directamente ligada a nuestra subjetividad.

La mención del principio de incertidumbre de Heisenberg por parte de Fernando González, sirve para ilustrar cómo los avances en la física cuántica han desafiado la noción de objetividad absoluta, Esto resalta cómo esta idea puede ser complicada, ya que nuestra percepción de la realidad está influenciada por nuestra propia acción y observación, lo que respalda la conexión entre subjetividad y objetividad.

Aquí Fernando González usa el trabajo de Vygotsky para argumentar que la subjetividad y las emociones humanas son fundamentales en la comprensión de los fenómenos psicológicos y sociales, Esto apoya la idea de que la subjetividad (cómo sentimos y pensamos) es crucial para comprender los fenómenos sociales, ya que nuestras emociones influyen directamente en cómo interpretamos y reaccionamos ante ellos.

La crítica al positivismo y la neutralidad destaca cómo estas posturas limitan la capacidad de los investigadores para abordar fenómenos sociales complejos. Al considerar la neutralidad como una forma de evitar la subjetividad, se argumenta que los investigadores deben adoptar una postura más reflexiva y crítica que reconozca y utilice su propia subjetividad como un recurso para una comprensión más rica y auténtica de los datos.

Objetividad y Subjetividad en la Investigación Social Objetividad: Tradicionalmente se entiende como la capacidad de observar y analizar fenómenos de manera imparcial, sin influencias personales o emocionales. Sin embargo, el texto sugiere que esta forma de objetividad es limitada, ya que no puede existir un conocimiento completamente independiente de los sentidos humanos.

Subjetividad: Reconoce que los individuos interpretan el mundo a través de sus propios sentidos, experiencias y emociones. Este enfoque valora las perspectivas individuales y contextuales, proporcionando una comprensión más rica y matizada de los fenómenos sociales.

Cuestionamientos sobre la Objetividad y Subjetividad: Definición de Objetividad:

Lenin: Define la materia como todo lo que existe independientemente de los sentidos. Crítica de FGR: FGR discrepa de esta definición, argumentando que el conocimiento no puede construirse independientemente de los sentidos, ya que estos participan en la construcción del saber. Implicación: La crítica sugiere que la objetividad absoluta, definida como una separación total de los sentidos, es inalcanzable e irrealista. Subjetividad como Objeto de las Ciencias Sociales:

Debate con Martin Packer: Packer plantea que es problemático considerar la subjetividad como objeto de estudio en las ciencias sociales debido a su oposición a la objetividad. Respuesta de FGR: FGR argumenta que no hay una oposición real entre subjetividad y objetividad en el pensamiento científico, y cuestiona cuál es la definición de objetividad que se está utilizando. Concreción de la Materialidad:

Lenin: Incluye lo subjetivo dentro de la materialidad, lo que sugiere que todo lo que existe, incluyendo las percepciones subjetivas, es material. FGR: Señala que, aunque las emociones y el mundo psicológico son tan reales como el mundo físico, la subjetividad no puede ser completamente objetivada o materializada. Influencia de Vygotsky:

Vygotsky: Reconoce que las emociones y el mundo psicológico son reales y se expresan en nuestras acciones y fantasías. FGR: Utiliza la afirmación de Vygotsky para subrayar que la subjetividad es una parte esencial de la realidad humana y que no puede ser ignorada en la investigación social. Relación entre Subjetividad y Cultura:

FGR: Argumenta que la cultura es una producción subjetiva y que la subjetividad y la cultura se configuran recíprocamente. Las producciones subjetivas definen nuevas formas de cultura y viceversa. Implicación: Este punto resalta que la subjetividad humana es fundamental para la creación y recreación de la cultura, lo que sugiere que cualquier estudio cultural debe considerar la subjetividad. Potencial Creativo de la Subjetividad:

FGR: La subjetividad humana permite un potencial creativo que no está definido únicamente por las condiciones objetivas en las que surge el creador. Implicación: Este argumento sugiere que la creatividad y la innovación están intrínsecamente ligadas a la subjetividad, desafiando la noción de que las condiciones objetivas son las únicas determinantes.

Objetividad y Subjetividad en la Investigación Social Objetividad: Tradicionalmente se entiende como la capacidad de observar y analizar fenómenos de manera imparcial, sin influencias personales o emocionales. Sin embargo, el texto sugiere que esta forma de objetividad es limitada, ya que no puede existir un conocimiento completamente independiente de los sentidos humanos.

Subjetividad: Reconoce que los individuos interpretan el mundo a través de sus propios sentidos, experiencias y emociones. Este enfoque valora las perspectivas individuales y contextuales, proporcionando una comprensión más rica y matizada de los fenómenos sociales.

El texto nos pone en evidencia que la objetividad en la investigación social no puede desligarse completamente de la subjetividad. Ambos conceptos están interrelacionados y son esenciales para una comprensión completa y profunda de la realidad social. La crítica a la definición de Lenin subraya la importancia de reconocer la influencia de los sentidos y las percepciones subjetivas en la construcción del conocimiento. La referencia a Vygotsky refuerza la idea de que nuestras ideas y conocimientos están impregnados de subjetividad, lo que debe ser considerado en cualquier investigación social.

Este argumento defiende que la ciencia debe ser vista como un proceso creativo y subjetivo, en lugar de una mera aplicación de métodos estandarizados. La subjetividad del investigador es crucial para desarrollar nuevas formas de comprensión.

El reconocimiento de que la cultura es producida subjetivamente y luego objetivada resalta cómo la objetividad y la subjetividad están interrelacionadas en la construcción del conocimiento. Este enfoque sugiere que los fenómenos sociales no pueden entenderse completamente sin considerar cómo las percepciones y las experiencias subjetivas influyen en la creación y el significado de las formas culturales y sociales.

hipótesis del texto segun mi criterio "La objetividad y la subjetividad en la investigación social no son mutuamente excluyentes; en cambio, su integración puede proporcionar una visión más holística y profunda de los fenómenos sociales."

La idea de que la subjetividad es una forma objetiva de los procesos humanos desafía la visión tradicional de que sólo la objetividad puede proporcionar conocimiento válido. Al reconocer la subjetividad como un componente esencial en el análisis de fenómenos sociales, se abre la posibilidad de una comprensión más profunda y matizada de la realidad, que incluye la perspectiva personal del investigador y las influencias emocionales y culturales en el proceso investigativo.

La epistemología cualitativa y el estudio de la subjetividad en una perspectiva histórico-cultural, son esenciales para obtener una visión profunda de las experiencias humanas, reconociendo la influencia de los contextos específicos en la construcción de significados.

Reconocer la influencia de la subjetividad en la investigación social es esencial, ya que la neutralidad completa es un mito. Los investigadores deben considerar cómo sus propias perspectivas afectan la interpretación de los datos para obtener una visión más auténtica.

La epistemología cualitativa valora lo singular como una fuente válida de conocimiento, enfocándose en cómo las experiencias únicas aportan insights profundos sobre los fenómenos sociales, en lugar de buscar generalizaciones universales.

Aunque la cultura se manifiesta objetivamente, su origen es subjetivo. Para entender los fenómenos sociales, es crucial considerar tanto las manifestaciones objetivas como las interpretaciones subjetivas de la cultura.

Reconocer la complejidad y la naturaleza dinámica de la investigación social permite a los investigadores adoptar enfoques más innovadores y sensibles a las realidades cambiantes, mejorando así la calidad y relevancia del conocimiento generado.

A la pregunta, ¿De qué forma los sujetos modifican la manera en la que se establece un fenómeno social? El texto señala que los sujetos modifican la manera en que se establece un fenómeno social a través de la interacción activa con el investigador. Este proceso transforma el fenómeno, ya que el investigador ajusta los instrumentos de investigación para fomentar un diálogo auténtico y dinámico. Así, la investigación se convierte en una co-creación entre el investigador y los sujetos, afectando cómo se configura y comprende el fenómeno social.

Fernando González Rey critica la obsolescencia de los programas de formación en psicología, que perpetúan una visión instrumental y positivista, resaltando una desconexión entre los métodos de enseñanza tradicionales y las necesidades actuales de una investigación más holística y subjetiva. La dependencia de métodos anticuados limita la capacidad de los investigadores para abordar problemas complejos con una perspectiva crítica y dinámica.

Subjetividad y Objetividad en la investigación Social: González Rey plantea una visión integradora donde la subjetividad no se opone a la objetividad, sino que la enriquece, considerando la subjetividad como un proceso objetivo, se refleja cómo los aspectos personales y culturales influyen en la construcción de saberes científicos.

La hipótesis del texto sostiene que la subjetividad es esencial en la investigación científica y que debe ser integrada de manera activa en el proceso investigativo. En lugar de seguir métodos rígidos, se aboga por un enfoque dinámico donde el investigador interactúe y ajuste su enfoque según las circunstancias y hallazgos. Este enfoque propone que la subjetividad del investigador enriquece el conocimiento, al considerarse como una parte legítima y objetiva del proceso científico, en lugar de ser vista como una interferencia.

González Rey enfatiza que la subjetividad, al ser una fuente de resistencia y creatividad, permite a los individuos desafiar y cambiar las narrativas sociales dominantes, lo cual modifica la forma en que se entienden y establecen los fenómenos sociales.

Se resalta cómo la subjetividad permite la creación de nuevas formas de entendimiento y resistencia frente a las estructuras de poder establecidas. Los sujetos, a través de sus interpretaciones y perspectivas personales, pueden modificar y cuestionar fenómenos sociales.

La falta de comprensión sobre comportamientos o fenómenos puede llevar a etiquetarlos como patológicos, lo cual refleja una falta de objetividad y una tendencia a simplificar la realidad para ajustarla a esquemas preexistentes.

González Rey argumenta que la subjetividad, entendida como la interpretación personal y cultural, influye en la forma en que los individuos se relacionan con fenómenos complejos. La objetividad se cuestiona cuando se simplifican fenómenos sociales complejos a través de interpretaciones que no toman en cuenta la dimensión subjetiva de los participantes.

Hipótesis del texto: La epistemología cualitativa se diferencia de la investigación cualitativa tradicional al centrarse en un enfoque constructivo-interpretativo del conocimiento, que no se basa en las expresiones explícitas y descriptivas de los individuos estudiados, sino en aspectos indirectos que permiten interpretaciones no evidentes, superando así las limitaciones del empirismo al teórico y ofreciendo una comprensión más profunda y efectiva de la subjetividad y los fenómenos humanos. Esta hipótesis se centra en la capacidad de la investigación cualitativa para captar las dimensiones subjetivas y objetivas contextuales de la realidad social, destacando la importancia de los significados y experiencias individuales en la comprensión de fenómenos sociales complejos.

González Rey cuestiona la noción de una objetividad absoluta, como la propuesta por Lenin, que considera que la materia existe independientemente de los sentidos. Según González Rey, esto no es viable porque nuestras percepciones y sentidos están involucrados en la construcción del conocimiento.

Objetividad y Subjetividad: La objetividad no puede ser separada de la subjetividad, ya que nuestras percepciones juegan un papel crucial en cómo entendemos y construimos la realidad.

La subjetividad: Es vista como un proceso vinculado a la historia, la cultura y los contextos sociales, esta definición amplia reconoce que la subjetividad no es solo una característica psíquica, sino una construcción dinámica que emerge en contextos culturales y sociales.

La subjetividad: Aquí se destaca la ambigüedad del concepto de subjetividad, que puede ser visto desde la perspectiva del sujeto racional cartesiano o como una amenaza para la objetividad científica, este conflicto subraya la dificultad de integrar la subjetividad en paradigmas que priorizan la objetividad y el conocimiento empírico.

La subjetividad, lejos de ser un elemento puramente subjetivo y, por lo tanto, menos objetivo, es en realidad una forma de producción altamente objetiva en el contexto humano. Esto implica que nuestras emociones, percepciones y experiencias subjetivas contribuyen a la creación de conocimiento y cultura de una manera que puede ser considerada objetiva y relevante para el entendimiento científico.

La subjetividad se presenta como una producción simbólica-emocional que permite a los humanos generar significados sobre el mundo que los rodea. Esto implica que la subjetividad es un fenómeno objetivo en el sentido de que todos los humanos, independientemente de su contexto, son generadores de subjetividad.

En la psicología soviética, la subjetividad estaba restringida debido a las implicaciones ideológicas del materialismo frente al idealismo, esto nos muestra cómo los contextos políticos y filosóficos pueden limitar o influir en el enfoque de las ciencias sociales. La ausencia de una discusión abierta sobre la subjetividad refleja una resistencia a integrar aspectos del sujeto que no encajaban con las doctrinas materialistas predominantes.

La personalidad fue una forma de abordar la subjetividad dentro de un marco aceptable en la psicología soviética, esto lleva a una adaptación dentro de las limitaciones ideológicas para explorar aspectos del sujeto que estaban más cerca de lo que se consideraba aceptable, como la motivación humana.

La obra de Vygotsky y Bozhovich representó avances importantes en la comprensión de la subjetividad. Vygotsky introdujo conceptos que integraban la subjetividad en el pensamiento y el desarrollo, mientras que Bozhovich amplió estas ideas al considerar la personalidad como un sistema complejo de formaciones psicológicas, reflejando un esfuerzo por integrar la subjetividad en un marco teórico más amplio y dinámico.

Modificación de Fenómenos Sociales por los Sujetos: Construcción Social de la Realidad: Los sujetos, a través de sus interacciones y prácticas cotidianas, construyen y modifican continuamente la realidad social. La forma en que interpretan y actúan en el mundo influye en la definición y percepción de los fenómenos sociales. Ejemplo: Las normas sociales y valores culturales son construidos y reforzados mediante la interacción social. Participación y Agencia: Los individuos tienen agencia, es decir, capacidad de actuar y tomar decisiones que afectan los fenómenos sociales. Sus acciones pueden desafiar, reproducir o transformar las estructuras sociales existentes. Ejemplo: Los movimientos sociales y políticos pueden surgir de la agencia de individuos y grupos que buscan cambiar las condiciones sociales. Subjetividad y Experiencia Personal: La subjetividad y las experiencias personales de los individuos influyen en cómo perciben y reaccionan ante los fenómenos sociales. Sus interpretaciones y respuestas pueden variar significativamente según su contexto y biografía. Ejemplo: La percepción de la justicia o la injusticia puede variar entre individuos según sus experiencias personales con el sistema legal. Innovación y Cambio Cultural: Los sujetos pueden introducir nuevas ideas, prácticas y tecnologías que transforman los fenómenos sociales. La innovación cultural y tecnológica es un proceso constante que redefine la realidad social. Ejemplo: El impacto de las redes sociales en la comunicación y la organización social ha cambiado profundamente las formas en que las personas interactúan y participan en la sociedad. Retroalimentación y Reflexividad: Los fenómenos sociales son reflexivos, es decir, los sujetos no solo participan en ellos, sino que también reflexionan sobre ellos y modifican sus comportamientos en respuesta a nuevos entendimientos y conocimientos. Ejemplo: La adopción de nuevas políticas públicas puede ser influenciada por la retroalimentación y el activismo de los ciudadanos.

Argumentos que Soportan el Texto: Argumentos Históricos y Filosóficos: Referencia a filósofos y psicólogos como Dilthey, Husserl, Heidegger y Gadamer para situar la discusión en un contexto histórico-filosófico. Crítica de la investigación cualitativa tradicional y su base en la fenomenología de Husserl, que buscaba una objetividad científica similar a la matemática. Argumentos Metodológicos: Crítica a la visión instrumental de la investigación cualitativa y cuantitativa que prevaleció en las ciencias sociales. Propuesta de una metodología constructiva-interpretativa que no se limita a las expresiones explícitas de los individuos, sino que busca interpretaciones profundas de los aspectos indirectos y simbólicos. Argumentos Empíricos y Teóricos: Ejemplos de cómo la investigación cualitativa basada en la Epistemología Cualitativa ha permitido comprender fenómenos complejos que otras teorías no lograban explicar. Uso de conceptos como "sentido subjetivo" y "formaciones psicológicas" para mostrar la aplicabilidad y profundidad de su enfoque teórico.

La discusión sobre objetividad y subjetividad en la investigación social es fundamental, ya que resalta la necesidad de un enfoque que reconozca la complejidad de la experiencia humana. Al integrar ambos aspectos, los investigadores pueden obtener una comprensión más completa de los fenómenos sociales, evitando reduccionismos que podrían limitar la interpretación de la realidad.

Objetividad y Subjetividad en la Investigación Social: Objetividad: El uso de métodos cuantitativos, como encuestas y experimentos controlados, que buscan obtener datos medibles y verificables. La replicabilidad de los estudios, donde los resultados pueden ser reproducidos por otros investigadores bajo las mismas condiciones. La minimización de la influencia del investigador en los resultados, mediante técnicas como el doble ciego. La adopción de una postura neutral y desapasionada para evitar sesgos personales. Subjetividad: La interpretación de datos cualitativos, como entrevistas, grupos focales y estudios de caso, que dependen en gran medida de la perspectiva del investigador. La construcción de significados y comprensiones basadas en la experiencia y el contexto de los participantes. La participación activa del investigador en el campo, que puede influir en la forma en que se recolectan y analizan los datos. La subjetividad como parte esencial del fenómeno estudiado, donde los significados y valores culturales son centrales.

La hipótesis central del texto que yo detecte es que la Epistemología Cualitativa, desarrollada por Fernando González Rey, ofrece una nueva perspectiva en la investigación de la subjetividad humana desde un enfoque cultural-histórico. Esta perspectiva difiere significativamente de otros enfoques cualitativos tradicionales y plantea una construcción del conocimiento que no se basa en la simple descripción inductiva de fenómenos observables, sino en una interpretación constructiva que incluye los aspectos subjetivos y simbólicos de la experiencia humana.

Considero que aquí puede estar la hipótesis:" La subjetividad y la objetividad no son opuestas en la investigación social, sino que se interrelacionan y coexisten, permitiendo una comprensión más profunda de los fenómenos sociales." Puesto que desafía la visión tradicional que considera la objetividad como un ideal a alcanzar en la investigación científica, sugiriendo que la subjetividad es igualmente válida y necesaria para entender la complejidad de la experiencia humana. Al reconocer que los investigadores y los sujetos de estudio aportan sus propias experiencias y contextos, se abre un espacio para una investigación más rica y matizada que puede captar la dinámica de los fenómenos sociales en su totalidad.

El texto argumenta que la subjetividad es un fenómeno objetivo en sí mismo, ya que es una producción simbólica-emocional de lo humano. Esto desafía la noción tradicional de que la subjetividad debe ser eliminada para alcanzar la objetividad en la investigación. En cambio, se propone que la subjetividad puede ofrecer una comprensión más profunda de los fenómenos sociales, siempre que se reconozca su contexto histórico y cultural .

Argumentos que soportan el texto Interacción Cultural: Se menciona que la subjetividad y la cultura se configuran de manera recíproca, lo que implica que las producciones subjetivas influyen en la cultura y viceversa .

Contribuciones de Vygotsky: Se hace referencia a conceptos de Vygotsky, como "perezhivanie" y "sentido", que apuntan a la importancia de la subjetividad en el desarrollo psicológico y social .

Cuantificación de la Subjetividad: Se argumenta que la mecánica cuántica ha desafiado la noción de objetividad en la ciencia, sugiriendo que la acción del observador es inseparable de los resultados de la investigación, lo que refuerza la idea de que la subjetividad es parte integral del conocimiento

La hipótesis central del texto podría ser que la investigación social debe integrar tanto la objetividad como la subjetividad para lograr una comprensión más completa y rica de los fenómenos sociales. Esto implica reconocer que la subjetividad no es un obstáculo para la objetividad, sino que puede ser una fuente legítima de conocimiento científico

Interacción entre Objetividad y Subjetividad: El texto sostiene que no hay una oposición estricta entre objetividad y subjetividad. En cambio, ambos conceptos pueden coexistir y complementarse en la investigación social. La subjetividad puede enriquecer la comprensión de fenómenos sociales al aportar perspectivas únicas y contextuales

Definición de Subjetividad: La subjetividad, por otro lado, se refiere a la experiencia personal y emocional de los individuos, que influye en su percepción y comprensión del mundo. Se argumenta que la subjetividad es una producción simbólica-emocional que es inherente a la condición humana y que se desarrolla en contextos culturales específicos

Definición de Objetividad: La objetividad se refiere a la capacidad de observar y analizar fenómenos sin la influencia de las emociones o prejuicios del investigador. En el contexto del texto, se menciona que la objetividad puede ser entendida de diferentes maneras, incluyendo la definición de Lenin sobre materia, que considera todo lo que existe independientemente de los sentidos

Definición de Objetividad: La objetividad se refiere a la capacidad de observar y analizar fenómenos sin la influencia de las emociones o prejuicios del investigador. En el contexto del texto, se menciona que la objetividad puede ser entendida de diferentes maneras, incluyendo la definición de Lenin sobre materia, que considera todo lo que existe independientemente de los sentidos

De acuerdo a Gonzáles Rey, argumenta que la teoría debe ser un sistema flexible y evaluado críticamente, en lugar de convertirse en un conjunto rígido de ideas dogmáticas. Las teorías deben estar en constante evolución y revisión para mantenerse relevantes y útiles en el avance del conocimiento.

En mi opinión, este texto destaca la importancia de reconocer cómo las omisiones y las limitaciones teóricas pueden afectar nuestra comprensión y estudio de los fenómenos sociales. Imagina que estás tratando de armar un rompecabezas, pero te faltan algunas piezas. Sin esas piezas, no puedes ver el cuadro completo y te perderías detalles importantes. Lo mismo ocurre en la teoría psicológica. Si no consideramos la subjetividad de las personas y cómo influye en su experiencia social, estamos dejando de lado una parte esencial del rompecabezas.

Al estudiar los fenómenos sociales, es crucial incluir la subjetividad como un elemento clave en la investigación. Este fragmento explica cómo los investigadores cualitativos crean signos interpretativos para comprender los elementos subjetivos como opiniones, creencias y significado personal que pueden no ser evidentes de inmediato.

Method of teaching empathy

establishing facts

quote via u/edward_slizzerhands

Stradavarius : violin : varnish :: West German Engineering : typewriter : cigarettes

https://www.reddit.com/r/typewriters/comments/1e8avi4/comment/le5yhzr/?utm_source=reddit&utm_medium=web2x&context=3

quote via u/A_newer_throwaway

There is a movie titled "Lucy" that is about a woman whose brain capacity increases from the "normal" 10%. As her brain capacity increases, she gains abilities, physical and intellectual, beyond what any other human can do. This movie runs on the myth that humans only use 10% of our brains, which is commonly believed by adults in America. I have included a link to a trailer, where Morgan Freeman can be heard telling a group of people that we only use 10% of our brains. https://youtu.be/MVt32qoyhi0?si=BT41UKro8DyDUAQT&t=71

The article I chose debunks the myth that we use only 10% of our brains. It states that we use our brains for our everyday life. How even clenching and unclenching our hands uses more than 10 percent of our brains.

https://www.bbc.com/future/article/20121112-do-we-only-use-10-of-our-brains

I've always been told that cognitive ability drops off as we age, so it was interesting to learn that there are areas that improve with age.

This article does a good job of talking about the effects of age on cognitive ability: https://onlinelibrary.wiley.com/doi/abs/10.1002/gps.1023?casa_token=uNi27yxoz6IAAAAA%3AIZMylwXOJE8swbHh8LMjU_DT722tQF-7v2REkID1k9dTq7ftDtMcb2xCGAotAA0hcn3b0TTAOvtfplM

Due to the teacher shortage, there are new ways to get your teaching certificate. All you have to have is a bachelors degree and take the alt education method to get certified. I understand the need for this because of the shortage however it does not value the years educators have put in to be a teacher.

I do feel the education system needs changed in some ways. Students academics are so diverse that we need to stop assessing students using the same test.

This has allowed Superintendents to communicate with staff and community. They also need to support their technology department to make sure students have the best technology to learn.

Having a Superintendent who communicates with all staff just not administrators. Staff feels as those they are not just a staff member but an actual individual. When times are heated having open communication really helps to keep turmoil down.

No one likes changes but education has definitely changed in the last 10-15 years. I have been out of school for 19 years and being in the school system things have definitely changed. Families, academics, community, and students have changed since I started teaching 12 years ago.

These change are most definitely impacting education and some is for the good and some is for the not so good. It seems as though politics are driving the force for education and they are not the ones who in the schools everyday.

My school district is definitely small and our community is everything. The Superintendent has been working on building connections with our community. The community used to be everything in our school district.

As I was reading this paragraph I was thinking how at times the Superintendent needs to act as if they are in a business role.

There are so many issues the Superintendent has to deal with on a daily that they do not get to be that master teacher.

I know there is a specific formula that state board of education uses for personnel. My school district is always over the state formula. It is usually in the special education department. We have many severe students who need more adult supervision. I am not sure how we could have less staff members.

Sounds right, but double check https://new.reddit.com/r/typewriters/comments/1e8r1vi/olivetti_dora/

Another example of this would be that black cats are bad luck. There is no relationship between the two and no hard evidence to back this up. It was believed to be true back in Europe around the middle ages, where witchcraft and associations with the devil were prevalent. People back then thought black cats were witches in disguise.

https://imgflip.com/i/4fgry7

https://www.magicmargin.net/2011/07/typewriter-cover-pattern.html

Sewing pattern for a typewriter cover

Looking at the learning objectives on the class website, I have skimmed through this chapter and found some figures and sections that I think will give me the most important information for understanding our nervous system, how it communicates, its structure, and how we assess brain activity. - Gene-Environment Interactions: Even though this doesn't necessarily fit one of the learning objectives, I think this is an important topic throughout this course. It is important to look at how our genes and our environment interact to make us who we are. -Neuronal Connection: I think this fits in with the learning objective about how the brain communicates with the body. I think Figure 3.13 can help with understanding this topic. - Brain Imaging: I think this fits in with the learning objective about assessing brain activity and function. - Forebrain Structures - Midbrain and Hindbrain Structures: I think these last two fit in with the learning objective about the basic structures of the brain and their primary functions. - Table 3.2 and Table 3.1 seem to give important information in an easy to understand way.

These lines describe the loss of sight. First, something gets between the narrator and the light. Then their eyes stop working. In the end, they can't see at all. The writer uses "Windows" to describe the eyes. The repetition of the word "see" emphasizes the importance of sight. The dashes make the writing feel jerky. This matches how suddenly losing sight might feel-- abrupt and incohesive.

YESS

swanns...

oh nvm

selmy?

the love one SCARES me

nightwalker name?

tf??

ahh so thats why the tyrells were in KL at the end of the season

i really don't like him

:((

Relación con Bifo

Being able to take data and covert it into tables makes it easy for presentations. It is nice to have digital data so you can filter and sort data quickly and easily.

This is a great point about working collaboratively with staff, parents, board members and community members. Unfortunately some students do not have the support and need that community support.

DOI: 10.1016/j.celrep.2021.108895

Resource: (Active Motif Cat# 39036, RRID:AB_2335600)

Curator: @Naa003

SciCrunch record: RRID:AB_2335600

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What is this?

DOI: 10.1016/j.celrep.2021.108875

Resource: (Thermo Fisher Scientific Cat# A32730, RRID:AB_2633279)

Curator: @Naa003

SciCrunch record: RRID:AB_2633279

---

What is this?

DOI: 10.3390/jdb9040043

Resource: (BDSC Cat# 35770,RRID:BDSC_35770)

Curator: @mpairish

SciCrunch record: RRID:BDSC_35770

---

What is this?

DOI: 10.3390/ijms222011153

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @mpairish

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.1016/j.isci.2021.103314

Resource: (BDSC Cat# 5905,RRID:BDSC_5905)

Curator: @mpairish

SciCrunch record: RRID:BDSC_5905

---

What is this?

DOI: 10.1073/pnas.2016878118

Resource: (BDSC Cat# 1495,RRID:BDSC_1495)

Curator: @bandrow

SciCrunch record: RRID:BDSC_1495

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What is this?

DOI: 10.3390/life11010067

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.3390/life11010067

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.17912/micropub.biology.000356

Resource: (BDSC Cat# 1495,RRID:BDSC_1495)

Curator: @bandrow

SciCrunch record: RRID:BDSC_1495

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What is this?

DOI: 10.1128/mBio.02205-20

Resource: (BDSC Cat# 31777,RRID:BDSC_31777)

Curator: @bandrow

SciCrunch record: RRID:BDSC_31777

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What is this?

DOI: 10.1128/mBio.02205-20

Resource: (BDSC Cat# 36111,RRID:BDSC_36111)

Curator: @bandrow

SciCrunch record: RRID:BDSC_36111

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What is this?

DOI: 10.1128/mBio.02205-20

Resource: (BDSC Cat# 58309,RRID:BDSC_58309)

Curator: @bandrow

SciCrunch record: RRID:BDSC_58309

---

What is this?

DOI: 10.1128/mBio.02205-20

Resource: (BDSC Cat# 44034,RRID:BDSC_44034)

Curator: @bandrow

SciCrunch record: RRID:BDSC_44034

---

What is this?

DOI: 10.1080/15476286.2020.1860580

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.1080/15476286.2020.1860580

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.1093/nar/gkaa1205

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.1093/nar/gkaa1205

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.1093/nar/gkaa1205

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.1093/nar/gkaa1205

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.1093/nar/gkaa1205

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.1093/nar/gkaa1205

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.1093/nar/gkaa1205

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.1093/nar/gkaa1205

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.1093/nar/gkaa1205

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.1093/nar/gkaa1205

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.1093/nar/gkaa1205

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.1093/nar/gkaa1205

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.1093/nar/gkaa1205

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.1007/s00439-020-02240-5

Resource: BDSC_15384

Curator: @bandrow

SciCrunch record: RRID:BDSC_15384

---

What is this?

DOI: 10.15252/embr.201949804

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.7554/eLife.72350

Resource: (BDSC Cat# 66542,RRID:BDSC_66542)

Curator: @mpairish

SciCrunch record: RRID:BDSC_66542

---

What is this?

DOI: 10.7554/eLife.72350

Resource: (BDSC Cat# 5823,RRID:BDSC_5823)

Curator: @mpairish

SciCrunch record: RRID:BDSC_5823

---

What is this?

DOI: 10.7554/eLife.72350

Resource: (BDSC Cat# 6595,RRID:BDSC_6595)

Curator: @mpairish

SciCrunch record: RRID:BDSC_6595

---

What is this?

DOI: 10.7554/eLife.72350

Resource: (BDSC Cat# 9468,RRID:BDSC_9468)

Curator: @mpairish

SciCrunch record: RRID:BDSC_9468

---

What is this?

DOI: 10.7554/eLife.72350

Resource: RRID:BDSC_5416

Curator: @mpairish

SciCrunch record: RRID:BDSC_5416

---

What is this?

DOI: 10.7554/eLife.72350

Resource: RRID:BDSC_675

Curator: @mpairish

SciCrunch record: RRID:BDSC_675

---

What is this?

DOI: 10.7554/eLife.72350

Resource: (BDSC Cat# 4590,RRID:BDSC_4590)

Curator: @mpairish

SciCrunch record: RRID:BDSC_4590

---

What is this?

DOI: 10.7554/eLife.72350

Resource: RRID:BDSC_27031

Curator: @mpairish

SciCrunch record: RRID:BDSC_27031

---

What is this?

DOI: 10.7554/eLife.72350

Resource: (BDSC Cat# 4775,RRID:BDSC_4775)

Curator: @mpairish

SciCrunch record: RRID:BDSC_4775

---

What is this?

DOI: 10.7554/eLife.72350

Resource: (BDSC Cat# 3605,RRID:BDSC_3605)

Curator: @mpairish

SciCrunch record: RRID:BDSC_3605

---

What is this?

DOI: 10.7554/eLife.72350

Resource: (BDSC Cat# 64349,RRID:BDSC_64349)

Curator: @mpairish

SciCrunch record: RRID:BDSC_64349

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What is this?

DOI: 10.1371/journal.pbio.3000689

Resource: BDSC_33985

Curator: @bandrow

SciCrunch record: RRID:BDSC_33985

---

What is this?

DOI: 10.1128/AEM.01401-20

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bandrow

SciCrunch record: RRID:SCR_006457

---

What is this?

DOI: 10.1098/rsob.200295

Resource: (BDSC Cat# 50422,RRID:BDSC_50422)

Curator: @bandrow

SciCrunch record: RRID:BDSC_50422

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What is this?

DOI: 10.1242/jeb.242699

Resource: BDSC_41803

Curator: @mpairish

SciCrunch record: RRID:BDSC_41803

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What is this?